Target gene segment for resisting root cancer, interference vector and application thereof
阅读说明：本技术 抗根癌病的靶基因片段及其干扰载体和应用 (Target gene segment for resisting root cancer, interference vector and application thereof ) 是由 齐希梁 李明 刘聪利 宋露露 于 2020-06-23 设计创作，主要内容包括：本发明公开了抗根癌病的靶基因片段及其干扰载体和应用。本发明通过病毒诱导的基因沉默技术将18段樱桃根瘤病菌的iaaM、iaaH和ipt的靶标片段及其基因融合靶标片段导入樱桃砧木‘ZY-1’中,发现导入18段靶基因片段的樱桃砧木的抗病性都显著提高,其中有5段靶标片段的抗病性极显著提高,达到高抗水平以上甚至免疫；本发明进一步将这5段靶基因序列构建稳定遗传干扰载体转化樱桃砧木,发现转基因樱桃植物对根癌土壤杆菌抗病性显著增强,达到高抗水平以上。应用本发明所提供的抗根癌病的靶基因片段能够提高果树对根癌病的抗性或进一步培育获得抗根癌病的果树新种质。(The invention discloses a target gene segment for resisting root cancer, an interference vector and application thereof. According to the invention, 18 target segments of iaaM, iaaH and ipt of cherry rhizobium and gene fusion target segments thereof are introduced into cherry rootstocks 'ZY-1' by a virus-induced gene silencing technology, and the disease resistance of the cherry rootstocks introduced with 18 target gene segments is remarkably improved, wherein the disease resistance of 5 target segments is remarkably improved, so that the effect of resisting water at a high level or above and even immunity is achieved; the invention further constructs a stable genetic interference vector for transforming the 5 target gene sequences into the cherry rootstocks, and finds that the disease resistance of the transgenic cherry plants to the agrobacterium tumefaciens is obviously enhanced to reach a high water level or above. The target gene segment for resisting the root cancer disease provided by the invention can improve the resistance of fruit trees to the root cancer disease or further culture and obtain new germplasm of the fruit trees for resisting the root cancer disease.)
1. A target gene fragment for root cancer resistance, wherein the target gene fragment is selected from any one of the target gene fragments of (1) to (4):
(1) any one of 4 target segments of iaaM gene, the 4 target segments of iaaM gene are iaaM-1, iaaM-2, iaaM-3 and iaaM-4, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in sequence;
(2) any one of 4 target segments of iaaH gene, the 4 target segments of iaaH gene are iaaH-1, iaaH-2, iaaH-3 and iaaH-4, and the nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 in sequence;
(3) any one of 4 target segments of the ipt gene, wherein the 4 target segments of the ipt gene are ipt-1, ipt-2, ipt-3 and ipt-4 respectively, and the nucleotide sequences of the 4 target segments of the ipt gene are shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 in sequence;
(4) the fusion target gene segment for resisting the root cancer is obtained by fusing target gene segments of any two genes of iaaM, iaaH or ipt genes, wherein the fusion target gene segments are any one of iaaM-ipt-1, iaaM-ipt-2, iaaM-iaaH-1, iaaM-iaaH-2, iaaH-ipt-1 or iaaH-ipt-2; wherein the nucleotide sequence of iaaM-ipt-1 is shown as SEQ ID NO.13, the nucleotide sequence of iaaM-ipt-2 is shown as SEQ ID NO.14, the nucleotide sequence of iaaM-iaaH-1 is shown as SEQ ID NO.15, the nucleotide sequence of iaaM-iaaH-2 is shown as SEQ ID NO.16, the nucleotide sequence of iaaH-ipt-1 is shown as SEQ ID NO.17, and the nucleotide sequence of iaaH-ipt-2 is shown as SEQ ID NO. 18.
2. The target gene fragment for root cancer resistance according to claim 1, wherein the target gene fragment is any one selected from iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1, and iaaM-ipt-2.
3. The target gene fragment for root cancer resistance according to claim 2, wherein the target gene fragment is iaaM-ipt-2.
4. RNA transcribed from the target gene segment for treating a root cancer according to any one of claims 1 to 3.
5. An RNA interference vector containing a target gene fragment against root cancer according to claims 1 to 3; preferably, the RNA interference vector is a Gateway interference vector.
6. A method of constructing the RNA interference vector of claim 5, comprising: and (3) inserting the target gene fragment for resisting the root cancer into a Gateway interference vector to obtain the gene fragment.
7. The method of claim 6, comprising: the target gene fragment is connected into pDONR207 through a BP reaction, and is constructed into pK7GWIWG2(I),0 through an LR reaction to obtain a Gateway interference vector.
8. A method of breeding fruit trees for resistance to agrobacterium tumefaciens, comprising: inserting the anti-root cancer target gene fragment of any one of claims 1 to 3 into a Gateway interference vector to construct an RNA interference vector; and transforming the constructed RNA interference vector into fruit tree cells or tissues.
9. A method for cultivating a new variety of fruit trees resistant to Agrobacterium tumefaciens comprises the following steps: (1) inserting the anti-root cancer target gene fragment of any one of claims 1 to 3 into a Gateway interference vector to construct an RNA interference vector; (2) transforming the constructed RNA interference vector into fruit tree cells or tissues; (3) screening to obtain a new variety of transgenic fruit trees with improved disease resistance to agrobacterium tumefaciens.
10. A method according to claim 8 or 9, wherein the fruit tree comprises cherries, peaches, plums, apricots or grapes.
Technical Field
The invention relates to target gene segments for resisting root cancer, further relates to an interference vector containing the target gene segments and application of the interference vector in improving the resistance of fruit trees to agrobacterium tumefaciens, and belongs to the field of target gene segments for resisting root cancer and application of the target gene segments.
Background
The European sweet cherry (Prunus avium. L) belongs to the fruit tree plants of Rosaceae, Prunoideae and Prunus, is one of the tree species with the best cultivation benefit in the northern deciduous fruit trees at present, and is the earliest fresh fruit on the market in northern spring of China. The cultivation area and the yield of the Chinese sweet cherries reach the first world at present. Although the Chinese sweet cherry industry achieves favorable performance, a plurality of problems still exist in production, such as cherry crown gall and the like, and seriously threaten the Chinese cherry industry (Zhang Kai Chun, and the like, the cultivation history, the current production situation and the development suggestion of Chinese sweet cherries, deciduous fruit trees, 2017,49(06): 1-5.).
Cherry crown gall, a bacterial disease caused by pathogenic bacteria in the genus Agrobacterium spp, infects a variety of higher plants (Maducong et al, fruit crown gall and biocontrol thereof, fruit tree, 1995,2(5): 42-44.). The agrobacterium tumefaciens can invade from a plant wound, under the condition that host tissues exist, the bacteria do not enter host plant cells, but a T-DNA fragment of Ti plasmid is introduced into the genome of the plant cells, and a plurality of key genes for inducing tumors, such as a tryptophan monooxygenase gene (iaaM) which is a key enzyme in auxin synthesis, an indoleacetamide hydrolase gene (iaaH) and an isopentenyl transferase gene (ipt) which is a key enzyme in cytokinin synthesis, are contained on the T-DNA. The disease has high incidence and high propagation speed, and can cause the disease in the whole garden when the disease is serious, thereby causing serious harm to the Chinese cherry industry (Gohlke J, Deeken R. Plant responses to Agrobacterium tumefaciens and crown grain reduction. frontiers in Plant Science 2014,5: 155.). Therefore, it is imperative to deeply research and effectively prevent and treat the root cancer threatening the development of the cherry industry. The selection of the disease-resistant variety or the rootstock and the improvement of the disease resistance of the rootstock are important ways for preventing and treating the cherry root cancer, and the breeding period of the disease-resistant variety or the rootstock is long, so that the improvement of the disease resistance of the existing rootstock variety by means of biotechnology and the like is a quick and effective means.
Host-induced gene silencing (HIGS) was developed on the basis of virus-induced gene silencing (VIGS), and is also a manifestation of post-transcriptional gene silencing (PTGS) -RNA silencing (Baulcombe DC. VIGS, HIGS and FIGS: small RNA silencing in the interaction of viruses or genes organization in Plant Biology 2015, 141-146.). The specific gene of the pathogenic bacteria is transfected into a host plant through a virus vector or a target gene fragment antisense hairpin structure vector containing double promoters, so that the pathogenic agent generates double-stranded RNA (dsRNA) with a specific sequence. When a pathogen attacks a host expressing a HIGS structure, a target gene in a pathogen is down-regulated, namely, the expression of an endogenous gene of the pathogen in a host plant is interfered by an RNAi structure, and finally, a gene of a plant pathogen is silenced (Nunes and Dean, 2012; Yin and Hulbert, 2018). HIGS, as a new technology with rapidness, simplicity, strong universality and high targeting, overcomes the defects of the traditional gene function research method, and shows strong advantages in the research of the gene function of plant obligate parasitic bacteria (Xiaoyao, and the like, the research and application progress of host-induced gene silencing technology, the plant protection academy, 2020,47(01): 11-17.).
Therefore, the target fragments of key auxin synthetic enzymes iaaM and iaaH and a key cytokinin synthetic enzyme ipt for resisting the root cancer disease or the fusion target fragments thereof are obtained by screening by using the HIGS technology, and the method has important values for improving the disease resistance of cherry rootstocks or breeding new varieties of fruit trees resisting the root cancer disease and the like.
Disclosure of Invention
It is an object of the present invention to provide a target gene fragment or a fusion target gene fragment for treating root cancer;
the other purpose of the invention is to provide an interference vector containing a target gene segment or a fusion target gene segment of the root cancer;
the third purpose of the invention is to apply the target gene segment or the fusion target and the interference vector containing the target gene segment to improve the disease resistance of fruit trees to the root cancer or construct new fruit tree germplasm for resisting the root cancer.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the present invention firstly provides a target gene fragment for resisting root cancer, wherein the target gene fragment is selected from any one of target gene fragments (1) to (3):
(1) any one of 4 target segments of iaaM gene, the 4 target segments of iaaM gene are iaaM-1, iaaM-2, iaaM-3 and iaaM-4, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in sequence;
(2) any one of 4 target segments of iaaH gene, the 4 target segments of iaaH gene are iaaH-1, iaaH-2, iaaH-3 and iaaH-4, and the nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 in sequence;
(3) any one of 4 target segments of the ipt gene, wherein the 4 target segments of the ipt gene are ipt-1, ipt-2, ipt-3 and ipt-4 respectively, and the nucleotide sequences of the target segments are shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 in sequence.
The transgenic cherry rootstock 'ZY-1' of iaaM-2, iaaM-4 or ipt-2 target fragments shows remarkable disease resistance to agrobacterium tumefaciens, and the plant reaches a high water-resistant level or above; thus, the target gene fragment against root cancer described in the present invention is preferably any of iaaM-2(SEQ ID NO.2), iaaM-4(SEQ ID NO.4), or ipt-2(SEQ ID NO.10) target fragments.
The invention also provides a fusion target gene segment for resisting the root cancer, which is obtained by fusing target gene segments of any two genes of iaaM, iaaH or ipt genes, wherein the fusion target gene segments are iaaM-ipt-1(SEQ ID NO.13), iaaM-ipt-2(SEQ ID NO.14), iaaM-iaaH-1(SEQ ID NO.15), iaaM-iaaH-2(SEQ ID NO.16), iaaH-ipt-1(SEQ ID NO.17) and iaaH-ipt-2(SEQ ID NO. 18); wherein the nucleotide sequence of iaaM-ipt-1 is shown as SEQ ID NO.13, the nucleotide sequence of iaaM-ipt-2 is shown as SEQ ID NO.14, the nucleotide sequence of iaaM-iaaH-1 is shown as SEQ ID NO.15, the nucleotide sequence of iaaM-iaaH-2 is shown as SEQ ID NO.16, the nucleotide sequence of iaaH-ipt-1 is shown as SEQ ID NO.17, and the nucleotide sequence of iaaH-ipt-2 is shown as SEQ ID NO. 18.
The method is characterized in that the transgenic cherry rootstock 'ZY-1' of two fused target segments, namely iaaM-ipt-1 and iaaM-ipt-2, shows remarkable disease resistance to agrobacterium tumefaciens, plants reach a high water resistance level or above, and particularly the transgenic cherry rootstock 'ZY-1' of the obtained iaaM-ipt-2 target segment has immune level to the disease resistance of agrobacterium tumefaciens; thus, the fusion target gene fragment against root cancer described in the present invention is preferably any of the fusion target fragments represented by iaaM-ipt-1(SEQ ID NO.13) or iaaM-ipt-2(SEQ ID NO. 14).
The invention further provides RNA transcribed by the anti-root cancer target gene segment or the fusion target gene segment.
The invention further provides an RNA interference vector containing the anti-root cancer target gene segment or the fusion target gene segment; preferably, the RNA interference vector is a Gateway interference vector.
The invention further provides a method for constructing the RNA interference vector, which comprises the following steps: inserting the target gene fragment or the fusion target gene fragment for resisting the root cancer into a Gateway interference vector to obtain an RNA interference vector; preferably, the target gene fragment or the fusion target gene fragment is connected to pDONR207 through a BP reaction, and is constructed into pK7GWIWG2(I),0 through an LR reaction to obtain the Gateway interference vector.
The invention provides a method for cultivating resistance of fruit trees to agrobacterium tumefaciens, which comprises the following steps: inserting the target gene segment or the fusion target gene segment for resisting the root cancer into a Gateway interference vector to obtain an RNA interference vector; and transforming the constructed RNA interference vector into fruit tree cells or tissues.
The invention further provides a method for cultivating a new variety of fruit trees resistant to agrobacterium tumefaciens, which comprises the following steps: (1) inserting the target gene segment or the fusion target gene segment for resisting the root cancer into a Gateway interference vector to obtain an RNA interference vector; (2) transforming the constructed RNA interference vector into fruit tree cells or tissues; (3) screening to obtain a new variety of transgenic fruit trees with improved disease resistance to agrobacterium tumefaciens.
The fruit trees can be various fruit trees such as cherry, peach, plum, apricot, grape and the like, and the cherry is preferred.
The protocol for such transformation, and the protocol for introducing the nucleotide into a plant, may vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the nucleotide into a plant cell include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and the like.
Detailed description of the invention
The invention introduces 18 target segments of iaaM, iaaH and ipt of cherry rhizobium and gene fusion target segments thereof into cherry rootstock 'ZY-1' by a virus-induced gene silencing technology (VIGS), instantly expresses dsRNA, and then performs Agrobacterium tumefaciens inoculation, and finds that the disease resistance of the cherry rootstock 'ZY-1' introduced with 18 target gene segments is obviously improved and reaches more than a disease resistance level, wherein the disease resistance of 5 target segments (or fusion segment sequences) is greatly improved, and reaches more than a high water resistance level and even immunity. Further, the research utilizes RNAi technology to construct a stable genetic interference vector of the 5 target gene sequences (reaching a high water resistance level or more and even immunity), and cherry rootstock ' ZY-1 ' is transformed to obtain a transgenic cherry rootstock ' ZY-1 plant, which has significantly enhanced disease resistance to Agrobacterium tumefaciens and reaches a high water resistance level or more, and particularly obtains a transgenic cherry rootstock ' ZY-1 ' with iaaM-ipt-2 target fragments, which has immunity level to Agrobacterium tumefaciens.
The target gene segment or the fusion target gene segment which is obtained by screening and resists the root cancer is used for constructing a stable genetic transformation vector and the genetic transformation vector is transferred into fruit trees such as cherry, peach, plum, apricot, grape and the like, so that the resistance of the fruit trees to the root cancer can be effectively improved, a new germplasm of the fruit trees which resists the root cancer can be expected to be cultivated, the control and the prevention of the root cancer of the fruit trees are realized, and the harm of the fruit trees in production is eliminated.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. probes 8:91-98 (1994)).
The term "recombinant host cell" or "host cell" means a cell comprising a nucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell. The host cell may be a prokaryotic cell or a eukaryotic cell.
The term "transformation" refers to a process of introducing a heterologous DNA sequence into a host cell or organism.
The term "expression" refers to the transcription and/or translation of an endogenous gene or transgene in a plant cell.
The term "RNA interference (RNAi)" means the phenomenon of inducing gene expression silencing of homologous sequences in cells by exogenous or endogenous double-stranded RNA.
Drawings
FIG. 1 crown gall diameter analysis of cherry rootstock 'ZY-1' transiently transformed with 18 segments of target gene. Indicates that there was a significant difference in t-test at p <0.01, and indicates that there was a very significant difference in t-test at p < 0.01.
FIG. 2. disease index analysis of cherry rootstock 'ZY-1' transiently transformed with 18 segments of target genes against Agrobacterium tumefaciens. Indicates that there was a significant difference in t-test at p <0.01, and indicates that there was a very significant difference in t-test at p < 0.01.
FIG. 3 genetic transformation System of cherry rootstock 'ZY-1'. (1) The cherry CAB-6p test-tube plantlet young leaf infected by the agrobacterium tumefaciens GV3101 is transferred to a regeneration culture medium to induce and regenerate the adventitious bud initial state (note: the leaf must be a closed young leaf, and the young leaf is scratched by a blade and is placed in agrobacterium liquid for infection). (2-3) the leaf blades were transferred to a growth state after 3 weeks in the regeneration induction medium. (4-5) inducing and regenerating adventitious buds after the leaves are transferred into a regeneration culture medium for 7 weeks. (6) And (4) transferring the leaf-induced regenerated plant into a rooting culture medium for 4 weeks to obtain the root.
FIG. 4 molecular characterization of genetically transformed plantlets of cherry rootstock 'ZY-1'. 1-3 (transgenic plants) electrophoresis channels are amplified to target gene segments corresponding to genes iaaM-2(A), iaaM-4(B), ipt-2(C), iaaM-ipt-1(D) and iaaM-ipt-2(E), Wt (wild plants) electrophoresis channels are not amplified to corresponding segments, plasmids (positive controls) can be amplified to corresponding target segments, and M is a DNA marker.
FIG. 5 evaluation of disease resistance of cherry rootstock 'ZY-1' transformed with target gene. A. Crown gall diameter statistical analysis. B. Disease index and disease resistance analysis. Indicates that there was a significant difference in t-test at p <0.01, and indicates that there was a very significant difference in t-test at p < 0.01.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Test example 1 screening of target fragments or fusion target fragments of key auxin synthesis enzymes iaaM and iaaH and key cytokinin synthesis enzyme ipt of Rhizoctonia cherry
1. Materials and methods
1.1 strains and vectors:
the Agrobacterium tumefaciens strain was isolated and purified from crown gall tissue of infected cherry trees in Miyauz county, Zhengzhou, Henan, and was identified as Agrobacterium tumefaciens (SEQ ID NO: HQ143621.1) by sequencing. Agrobacterium rhizogenes competence C58C1 was purchased from Shanghai Tokyo Biotechnology Ltd.
The carriers TRV1 and TRV2 are the gifts of doctor Liuyule of Qinghua university; the vectors pDONR207, pK7GWIWG2(I),0 were stored in the inventors' laboratory.
1.2 construction of VIGS-iaaM, VIGS-iaaH and VIGS-ipt transient interference vectors and inoculation of Agrobacterium rhizogenes and Agrobacterium tumefaciens.
Using total RNA of cherry rhizobium as a template, designing target gene primers of 4 pairs of iaaM (NP-059676.1) genes, 4 pairs of iaaH (NC-002377.1) genes and 4 pairs of ipt (YP-001967412.1) genes (the primer sequences are shown in Table 1):
TABLE 1 primer name and sequence information
Amplifying 4 target segment information of iaaM, iaaH and ipt genes respectively, wherein the length is 200-450bp, and the 4 target segments of the iaaM genes are iaaM-1(SEQ ID NO.1), iaaM-2(SEQ ID NO.2), iaaM-3(SEQ ID NO.3) and iaaM-4(SEQ ID NO.4) respectively; the 4 target segments of iaaH gene are iaaH-1(SEQ ID NO.5), iaaH-2(SEQ ID NO.6), iaaH-3(SEQ ID NO.7) and iaaH-4(SEQ ID NO.8) respectively; the 4 target sections of the ipt gene are ipt-1(SEQ ID NO.9), ipt-2(SEQ ID NO.10), ipt-3(SEQ ID NO.11) and ipt-4(SEQ ID NO.12) respectively; meanwhile, the fusion target fragments of 6 sections of iaaM, iaaH and ipt genes are respectively synthesized by utilizing a gene synthesis technology, the length is 200-450bp, and the 6 sections of fusion target fragments are iaaM-ipt-1(SEQ ID NO.13), iaaM-ipt-2(SEQ ID NO.14), iaaM-iaaH-1(SEQ ID NO.15), iaaM-iaaH-2(SEQ ID NO.16), iaaH-ipt-1(SEQ ID NO.17) and iaaH-ipt-2(SEQ ID NO. 18).
Amplifying 12 target fragments of iaaM, iaaH and ipt genes respectively and obtaining 6 fusion fragments through gene synthesis technology to be respectively connected to the multiple cloning site region of the carrier TRV2 (figure 1), obtaining recombinant carriers TRV2-iaaM-1, TRV2-iaaM-2, TRV2-iaaM-3, TRV2-iaaM-4, TRV2-iaaH-1, TRV2-iaaH-2, TRV2-iaaH-3, TRV2-iaaH-4, TRV2-ipt-1, TRV2-ipt-2, TRV2-ipt-3, TRV2-ipt-4, TRV2-iaaM-ipt-1, TRV2-iaaM-ipt-2, TRV2-iaaH-1, TRV 2-ipt-2-ipt-2, TRV-iaaM-6858-iaaH-1 and TRV-iaH-2-4, after the sequencing is correct, agrobacterium rhizogenes C58C1 is transformed by electric shock, and the sequence is compared with agrobacterium rhizogenes C58C1 strain 1 of TRV1 (carrying other part of gene information of TRV virus): 1 mixing, OD600And (3) injecting the mixed agrobacterium into cherry rootstock 'ZY-1' tender roots of 2-year-old potted cutting seedlings by using an injector, injecting at least more than 50 seedlings into each target segment, and performing at least 3 times of biological repetition. After 14 days, OD was used600Fresh Agrobacterium tumefaciens (Agrobacterium tumefaciens) (SEQ ID NO: HQ143621.1) suspension at a concentration of 0.8 was passed through a young-tip needleThe cherry rootstock ZY-1' generating the dsRNA segment is artificially inoculated by a thorn injection method. After 2 months, the disease condition of the cherry rootstock ZY-1' is observed, and the disease index is analyzed.
1.3 construction of Stable interference vectors
The invention selects 5 target gene segments iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1 and iaaM-ipt-2 with remarkable disease resistance by using Gateway technology. Primers containing BP sites at both ends (primer sequences are shown in Table 1) were designed, and attB1 site partial sequence (aaaaaagcaggct) was added to the 5 'end and attB2 site sequence (aagaaagctgggt) was added to the 3' end. And (3) respectively amplifying 5 target gene fragments with complete attB sites by using the TRV2 vector containing the target fragment as a template, cloning the target gene fragments to a pEASY-T1 vector, and constructing an RNAi vector by using a Gateway method. Two reactions take place for the construction of interfering expression vectors: (1) BP reaction: adopting an entry vector pDONRTM207 BP reaction refer to INVitrogn
1.4 genetic transformation and molecular identification of cherry rootstock' ZY-1
Using the group of the present inventorsAn established complete genetic transformation system of the agrobacterium tumefaciens mediated cherries (high-leaf happiness, etc., optimization of a regeneration system of isolated leaves of cherry rootstock CAB-6 p. fruit tree academy, 2008(04): 589. 592+623.), and a transgenic test material is a European sour cherry 'ZY-1' and a semi-wild cherry variety. Taking out tender and completely undeployed leaves from cherry test-tube seedlings with the age of 1 month for cutting, transversely cutting 5 knives with a scalpel perpendicular to the main vein of the leaves, and cutting off the main vein but not the leaves. Cutting the explants at OD600Soaking in 0.8-1.0 Agrobacterium solution for 5min, drying the bacterial solution on the surface of plant material with sterile filter paper, co-culturing the small leaves in WPM +6-BA 2mg/L + IAA 2mg/L + AS 20mg/L medium with filter paper bud differentiation, and dark culturing at 25 deg.C for 3 days. And transferring the co-cultured cherry explants to a resistant bud screening culture medium (WPM +6-BA 2mg/L + IAA 2mg/L + Cef 400mg/L + Kan 50mg/L) containing antibiotics for culture, wherein the illumination period is 16h of illumination/8 h of darkness, and buds can be generated after 6-8 weeks. When the resistant bud grows to 1-2 cm high, cutting off the bud and transferring the bud into a rooting culture medium (1/2MS + NAA 0.5mg/L + Kan 50mg/L) to induce rooting, and forming adventitious roots after 2-3 weeks.
After the obtained cherry rootstock ZY-1' is transformed into a strain, the strain is subjected to molecular identification. Extracting the total genome of the transgenic seedling as a template, designing iaaM-2-J-F/R, iaaM-4-J-F/R, ipt-2-J-F/R, iaaM-ipt-1-J-F/R, iaaM-ipt-2-J-F/R primers (the sequence is shown in table 1), carrying out PCR molecular detection, and carrying out subculture on the plant detected as the positive seedling to prepare for the next disease resistance experiment.
1.5 evaluation of resistance
The disease status of individual plants was classified into 5 grades according to the tumor diameter of the plants into which no interfering target fragments were introduced: grade 0 (immune): no disease, grade 1 (high resistance): the tumor diameter is 1.0-3.0 mm, and the grade 2 (disease resistance): the tumor diameter is 3.1-5.0 mm, and the grade 3 (susceptible diseases) is as follows: the tumor diameter is 5.1-7.0 mm, and the grade 4 (high sense): the diameter of the tumor is greater than 7.1 mm.
The resistance of the transgenic line of the cherry rootstock ZY-1' to the root cancer is classified into 5 grades according to the disease index. Immunity (R0), disease index 0; high disease resistance (HR), disease index 0-15; disease resistance (MR), disease index 15-30; infection (MS), disease index 30-45; high susceptibility (HM) and disease index of above 45.
Disease index ∑ (disease grade x number of strains with same disease grade)/highest disease grade x number of total strains investigated × 100
More than 60 strains per clonal material are inoculated per time and at least three biological replicates are performed.
1.6 data processing
The obtained data were processed and plotted using Microsoft Excel 2010 software; correlation analysis and significance of difference (p <0.05) analysis were performed using SPSS17.0 software.
2. Test results
Disease resistance analysis of VIGS-iaaM, VIGS-iaaH, VIGS-ipt and fusion target fragment instantaneously interfered with cherry rootstock' ZY-1
The
The analysis result shows that: the disease-sensitive symptoms (the existence of crown gall tumors and the diameter of the crown gall tumors) of the cherry rootstock 'ZY-1' introduced with the target gene segment and generating dsRNA to the agrobacterium tumefaciens are obviously lower than that of the cherry rootstock 'ZY-1' injected in an empty load manner. Inoculating the strain for 2 months, wherein the cherry rootstock ZY-1 introduced with the target gene segment has no obvious disease symptoms, only has individual crown gall and small diameter, and even has no crown gall; while the cherry rootstock 'ZY-1' injected with no load showed obvious symptoms of infection, marked crown gall and large diameter (FIG. 1). The test result shows that the cherry rootstock 'ZY-1' introduced with the target fragments of iaaM, iaaH and ipt and the fusion fragment of iaaM, iaaH and ipt genes has enhanced disease resistance to the agrobacterium tumefaciens.
The result of statistical analysis on the disease index of cherry rootstock ZY-1' inoculated with agrobacterium tumefaciens in the test shows that: after 2 months and 3 months of inoculation of agrobacterium tumefaciens, the disease indexes of the cherry rootstocks 'ZY-1' into which the target fragments of iaaM, iaaH and ipt and the fusion target fragments of iaaM, iaaH and ipt genes are introduced are 0% -19.36% and 0% -29.69%, respectively, while the disease indexes of the cherry rootstocks 'ZY-1' into which the target fragments are not introduced are 59.98% and 86.67%, respectively, indicating that the disease index of the cherry rootstocks 'ZY-1' into which the target fragments are introduced is significantly lower than that of the cherry rootstocks 'ZY-1' into which empty vectors are injected. Evaluation was performed according to the resistance classification criteria, and the cherry rootstocks ` ZY-1 ` introduced with the target gene fragments iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1 and iaaM-ipt-2 were highly resistant (immunized), the cherry rootstocks ` ZY-1 ` introduced with the target gene fragments iaaM-3, ipt-1, iaaM-iaaH-1, iaaH-ipt-1 and iaaH-ipt-2 were highly resistant, and the cherry rootstocks ` ZY-1 ` introduced with the target gene fragments iaaM-1, iaaH-2, iaaH-3, iaaH-4, ipt-3, ipt-4 and iaaM-iaaH-2 were disease resistant (FIG. 2).
Researches show that the disease resistance of cherry rootstock 'ZY-1' to agrobacterium tumefaciens is different when different gene target segments among 3 key genes synthesized by different auxins or cytokinins, namely iaaM, iaaH and ipt, are introduced, and the disease resistance of target fragments of the iaaM and ipt genes and fusion fragments thereof is remarkably higher than that of the target fragments of the iaaH genes and the fusion fragments thereof, so that the iaaM and the ipt are presumed to play important roles in the formation of crown gall tumor of agrobacterium tumefaciens.
The test results show that the cherry rootstock 'ZY-1' with the target segments of the 18 segments of iaaM, iaaH and ipt and the fusion segment of the iaaM, the iaaH and the ipt which are designed by the invention are instantaneously interfered has obviously enhanced disease resistance to the agrobacterium tumefaciens, and the plant shows disease resistance; meanwhile, the disease resistance of 18 different target gene fragments is different. In a word, the experiment obtains iaaM, iaaH and ipt gene target fragments and fusion target gene fragments which can improve the root cancer resistance of cherry rootstock 'ZY-1' through HIGS technology screening.
2.2 obtaining of transgenic tobacco plants
Through stable genetic transformation of the cherry rootstocks 'ZY-1' mediated by agrobacterium, interference vectors containing target fragments of iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1 and iaaM-ipt-2 are respectively transformed into the cherry rootstocks 'ZY-1', and the transgenic cherry rootstocks 'ZY-1' (figure 3) are finally obtained through kanamycin screening. PCR detection is carried out on the obtained transgenic cherry rootstock 'ZY-1', each target gene obtains a plurality of different positive transformation strains, and 3 transgenic strains are randomly selected respectively for next disease resistance analysis (figure 4).
2.3 evaluation of disease resistance of cherry rootstock 'ZY-1' transformed with target Gene
In order to determine whether the resistance level of the cherry rootstock 'ZY-1' strain of the target gene segment to agrobacterium tumefaciens can reach the immunity level or not, the experiment carries out the root cancer resistance identification on the cherry rootstock 'ZY-1' tissue culture seedlings of the target segments of iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1 and iaaM-ipt-2. The results show that: after 20 days of inoculation, white bulges grow on the inoculated part continuously by negative control (empty vector) without obtaining a target fragment, crown gall tumors begin to form, only partial plants of the cherry rootstock 'ZY-1' tissue culture seedlings which obtain iaaM-2, iaaM-4, iaaM-ipt-1 or ipt-2 target genes grow the white bulges, and the cherry rootstock 'ZY-1' which obtains the iaaM-ipt-2 target genes does not grow the white bulges on the inoculated part; after 40 days of inoculation, crown gall of negative control (transferred empty vector) of the target fragment did not develop and became larger in diameter, while only a few individual cherry rootstock 'ZY-1' tissue culture seedlings which obtained iaaM-2, iaaM-4, iaaM-ipt-1 or ipt-2 target genes formed crown gall, the crown gall diameter was significantly smaller than the negative control of the target fragment which was not obtained, and none of the cherry rootstock 'ZY-1' which obtained the iaaM-ipt-2 target gene still grew white protrusions at the inoculation site (FIG. 5A). Disease index analysis is carried out on the transgenic strain, and the results show that: after 2 months of inoculation, the disease indices of the cherry rootstock 'ZY-1' tissue culture seedlings for obtaining the target fragments of iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1 and iaaM-ipt-2 to Agrobacterium tumefaciens were 5.82%, 9.78%, 2.98%, 4.67% and 0%, respectively, while the disease index of the cherry rootstock 'ZY-1' tissue culture seedlings for not obtaining the negative control (transferred empty vector) of the target fragments to Agrobacterium tumefaciens was 68.90% (FIG. 6). The evaluation of the resistance classification criteria revealed that the disease resistance of the cherry rootstock 'ZY-1' tissue culture plantlets with the target fragments of iaaM-2, iaaM-4, ipt-2, iaaM-ipt-1 and iaaM-ipt-2 against Agrobacterium tumefaciens was significantly improved compared to the negative control (transferred vector) without the target fragments, the cherry rootstock 'ZY-1' with the target fragments of iaaM-2, iaaM-4, ipt-2 and iaaM-ipt-1 was of high resistance, and the cherry rootstock 'ZY-1' with the target fragment of iaaM-ipt-2 was of immune grade (FIG. 5B). The results show that: the transgenic cherry rootstock 'ZY-1' for obtaining iaaM-2, iaaM-4, iaaM-ipt-1, iaaM-ipt-2 and ipt-2 target segments shows remarkable disease resistance to agrobacterium tumefaciens, plants reach a high water level or above, and particularly the transgenic cherry rootstock 'ZY-1' for obtaining the iaaM-ipt-2 target segments has immune level to the disease resistance to agrobacterium tumefaciens.
SEQUENCE LISTING
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> target gene fragment for resisting root cancer, interference vector and application thereof
<130>HN-2002-200513A
<160>18
<170>PatentIn version 3.5
<210>1
<211>313
<212>DNA
<213>Agrobacterium spp
<400>1
gtttattgag atcctccgct tggtcatcaa cggatatgaa gaaaatcagc ggatgtgccc 60
tgaaggaatc tcagaacttc cacgtcggat cgcatctgaagtggttaacg gtgtgtctgt 120
gagccagcgc atatgccatg ttcaagtcag ggcgattcag aaggaaaaga caaaaataaa 180
gataaggctt aagagcggga tatctgaact ttatgataag gtggtggtca catctggact 240
cgcaaatatc caactcaggc attgcctgac atgcgatacc aatatttttc aggcaccagt 300
gaaccaagcg gtt 313
<210>2
<211>373
<212>DNA
<213>Agrobacterium spp
<400>2
cccaaatccc ggcacagtcg acacttactt ggtctaccaa ggcgtccaat acatgtggaa 60
agccgggcag ctgccaccga agctgttcca tcgcgtttac aacggttggc gtgcgttctt 120
gaaggacggt tttcatgagc gagatattgt gttggcttcg cctgtcgcta ttactcaggc 180
cttgaaatca ggagacatta ggtgggctca tgactcctgg caaatttggc tgaaccgttt 240
cgggagggag tccttctctt cagggataga gaggatcttt ctgggcacac atcctcctgg 300
tggtgaaaca tggagttttc ctcatgattg ggacctattc aagctaatgg gaataggatc 360
tggcgggttt ggt 373
<210>3
<211>435
<212>DNA
<213>Agrobacterium spp
<400>3
ggcgcgggca acagtgagtg gtctcgttgc catcgacttg gcaccatttt gcatggattt 60
ctccgaagca caactaatcc aagccctgtt tttgctgagc ggtaaaagat gtgcaccgat 120
tgatcttagt catttcgtgg ccatttcaat ctctaagact gccggctttc gaaccctgcc 180
aatgccgctg tacgagaatg gcacgatgaa atgcgttacc gggtttacca taacccttga 240
aggggccgtg ccatttgaca tggtagctta tggtcgaaac ctgatgctga agggttcggc 300
aggttccttt ccaacaatcg acttgctcta cgactacaga ccgttttttg accaatgttc 360
cgatagtgga cggatcggct tctttccgga ggatgttcct aagccgaaag tggcggtcat 420
tggcgctggc atttc 435
<210>4
<211>265
<212>DNA
<213>Agrobacterium spp
<400>4
gacgggatcg caaaagcagt gtattgcctg gactatgagt cgcaggatcc gaatggtaaa 60
ggtctagtgc tcatcagtta tacatgggag gacgactccc acaagctgtt ggcggtcccc 120
gacaaaaaag agcgattatg tctgctgcgg gacgcaattt cgagatcttt cccggcgttt 180
gcccagcacc tatttcctgc ctgcgctgat tacgaccaaa atgttattca acatgattgg 240
cttacagacg agaatgccgg gggag 265
<210>5
<211>309
<212>DNA
<213>Agrobacterium spp
<400>5
cagtcattcg tatcacacga cgtgattgct gaattcccac caataatggc gcaagctggg 60
ttcaagcttg gtatatttat ttggtctgaa tgggtttgaa atttccaact cagagagatg 120
gtggccatta cctcgttagc ccaaagccta gaacacctga aacggaaaga ctactcctgc 180
ttagaactag tagaaactct gatagcgcgt tgtgaagctg caaaatcatt aaacgccctt 240
ctggctacag actgggatgg tttgcggcga agcgccaaaa aaattgatcg ccatggaaac 300
gccggagta 309
<210>6
<211>342
<212>DNA
<213>Agrobacterium spp
<400>6
aggcgaacat cgctaccggc gtatttccca caagcgccgc tacgccggcg ctgataaacc 60
acttgccaaa gataccatcc cgcgtcgcag aaagactttt ttcagctgga gcactgccgg 120
gtgcctcggg aaatatgcat gagttatcgt ttggaattac aagcaacaac tatgccaccg 180
gggcggtgcg aaacccgtgg aatccagatc tgataccagg gggctcaagc ggtggtgtgg 240
ctgctgcggt agcaagccga ttgatgttag gcggcatagg caccgatacc ggtgcatctg 300
ttcgcctacc cgcagccctg tgtggcgtag taggatttcg ac 342
<210>7
<211>357
<212>DNA
<213>Agrobacterium spp
<400>7
gcagtgcgta gccgatgttg taatcctcga ccggataatt tccggcacac cggagagaat 60
accacccgtg ccgctgaagg ggctaaggat cggcctccct acaacctact tttatgatga 120
ccttgatgct gatgtggccc tagcagctga aacaacgatt cgcctgctag caaacaaagg 180
cgtaactttt gttgaagcta acattcccca ccttgacgaa ctgaataaag gggccagctt 240
cccagttgca ctctatgaat ttccacacgc tctaaaacag tatctcgacg actttgtaaa300
aactgtttct ttttctgacg tcatcaaagg aattcgtagc cctgatgtag ccaacat 357
<210>8
<211>357
<212>DNA
<213>Agrobacterium spp
<400>8
agatgctatt ctcttcccaa cagcaccctt ggtggccaga cccataggtc aggattcctc 60
agttatccac aatggcacga tgctggacac attcaagatc tacgtgcgaa atgtggaccc 120
aagcagcaac gcaggcctac ctggcttgag cattcctgtt tgcctgacac ctgatcgctt 180
gcctgttgga atggagatcg atggattagc ggattcagac caacgtctgt tagcaatcgg 240
gggggcattg gaagaagcca ttggattccg atattttgcc ggtttaccca attaaacttt 300
ctaccatgtt cgtttttaca atttttcaga ttgatgacaa tcaatccttg tattgcg 357
<210>9
<211>216
<212>DNA
<213>Agrobacterium spp
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aggagacagc catgccccac actttgttga aaaacaagtt gccttttggg aagaacctaa 60
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cttctcttgt gcaaaaaaat caatttgtat tcaacatatc gcaagaccga tggatctacg 180
tctaattttc ggtccaactt gcacaggaaa gacatc 216
<210>10
<211>248
<212>DNA
<213>Agrobacterium spp
<400>10
cgggcttatt cttgagggag gatctatctc gttgctcagg tgcatggcgc aaagtcgtta 60
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catgagcgtg gccaagacca gagttaagca gatgttacgc ccctctgcag gtctttctat 180
tatccaagag ttggttcaac tttggaggga gcctcggctg aggcccatac tggaagggat 240
cgatggat 248
<210>11
<211>235
<212>DNA
<213>Agrobacterium spp
<400>11
gaaagacatc gactgcgata gctcttgccc agcagactgg cctcccagtc ctctcgctcg 60
atcgcgtcca atgctgtcct caactatcaa ccggaagcgg gcgaccaaca gtggaagaac 120
tgaaaggaac gactcgtctg taccttgatg atcgcccttt ggtaaagggt atcattacag 180
ccaagcaagc tcatgaacgg ctcattgcgg aggtgcacaa tcacgaggcc aaagg 235
<210>12
<211>225
<212>DNA
<213>Agrobacterium spp
<400>12
aggcccatac tggaagggat cgatggatat cgatatgccc tgctatttgc tacccagaac 60
cagatcacgc ccgatatgct attgcagctc gacgcagata tggagaataa attgattcac 120
ggtatcgctc aggagtttct aatccatgcg cgtcgacagg aacagaaatt ccctttggtg 180
ggcgcgacag ctgtcgaagc gtttgaagga ccaccatttc gaatg 225
<210>13
<211>408
<212>DNA
<213>Artifical sequence
<400>13
ccctgttttt gctgagcggt aaaagatgtg caccgattga tcttagtcat ttcgtggcca 60
tttcaatctc taagactgcc ggctttcgaa ccctgccaat gccgctgtac gagaatggca 120
cgatgaaatg cgttaccggg tttaccataa cccttgaagg ggccgtgcca tttgacatgg 180
tagcttatgg tcgaaacctg atgctgccac ttgctcttca aggaggaata tcgaggaaga 240
gaatataaca gcctctggta cagacttctc ttgtgcaaaa aaatcaattt gtattcaaca 300
tatcgcaaga ccgatggatc tacgtctaat tttcggtcca acttgcacag gaaagacatc 360
gactgcgata gctcttgccc agcagactgg cctcccagtc ctctcgct 408
<210>14
<211>447
<212>DNA
<213>Artifical sequence
<400>14
cggttggcgt gcgttcttga aggacggttt tcatgagcga gatattgtgt tggcttcgcc 60
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aatttggctg aaccgtttcg ggagggagtc cttctcttca gggatagaga ggatctttct 180
gggcacacat cctcctggtg gtgtcgttgc tcaggtgcat ggcgcaaagt cgttattgga 240
acgcggattt tcgttggcat attattcgca acgagttagc agacgaggag agcttcatga 300
gcgtggccaa gaccagagtt aagcagatgt tacgcccctc tgcaggtctt tctattatcc 360
aagagttggt tcaactttgg agggagcctc ggctgaggcc catactggaa gggatcgatg 420
gatatcgata tgccctgcta tttgcta 447
<210>15
<211>448
<212>DNA
<213>Artifical sequence
<400>15
caggatccga atggtaaagg tctagtgctc atcagttata catgggagga cgactcccac 60
aagctgttgg cggtccccga caaaaaagag cgattatgtc tgctgcggga cgcaatttcg 120
agatctttcc cggcgtttgc ccagcaccta tttcctgcct gcgctgatta cgaccaaaat 180
gttattcaac atgattggct tacagacgag aatgccggcc ggggcggtgc gaaacccgtg 240
gaatccagat ctgataccag ggggctcaag cggtggtgtg gctgctgcgg tagcaagccg 300
attgatgtta ggcggcatag gcaccgatac cggtgcatct gttcgcctac ccgcagccct 360
gtgtggcgta gtaggatttc gaccgacgct tggtagatat ccgggagatc ggataatacc 420
ggttagccct acccgggaca ctcccgga 448
<210>16
<211>446
<212>DNA
<213>Artifical sequence
<400>16
acggttggcg tgcgttcttg aaggacggtt ttcatgagcg agatattgtg ttggcttcgc 60
ctgtcgctat tactcaggcc ttgaaatcag gagacattag gtgggctcat gactcctggc 120
aaatttggct gaaccgtttc gggagggagt ccttctcttc agggatagag aggatctttc 180
tgggcacaca tcctcctggt ggtgaaacat gcacacgctc taaaacagta tctcgacgac 240
tttgtaaaaa ctgtttcttt ttctgacgtc atcaaaggaa ttcgtagccc tgatgtagcc 300
aacattgcca atgcgcaaat tgatggacat caaatttcca aagctgaata tgaactggcc 360
cgccactcct tcagaccaag acttcaagcc acctatcgca actacttcaa actgaataga 420
ttagatgcta ttctcttccc aacagc 446
<210>17
<211>421
<212>DNA
<213>Artifical sequence
<400>17
aagactactc ctgcttagaa ctagtagaaa ctctgatagc gcgttgtgaa gctgcaaaat 60
cattaaacgc ccttctggct acagactggg atggtttgcg gcgaagcgcc aaaaaaattg 120
atcgccatgg aaacgccgga gtaggtcttt gcggcattcc actctgtttt aaggcgaaca 180
tcgctaccgg cgtaatgtta cgcccctctg caggtctttc tattatccaa gagttggttc 240
aactttggag ggagcctcgg ctgaggccca tactggaagg gatcgatgga tatcgatatg 300
ccctgctatt tgctacccag aaccagatca cgcccgatat gctattgcag ctcgacgcag 360
atatggagaa taaattgatt cacggtatcg ctcaggagtt tctaatccat gcgcgtcgac 420
a 421
<210>18
<211>445
<212>DNA
<213>Artifical sequence
<400>18
gccagcttcc cagttgcact ctatgaattt ccacacgctc taaaacagta tctcgacgac 60
tttgtaaaaa ctgtttcttt ttctgacgtc atcaaaggaa ttcgtagccc tgatgtagcc 120
aacattgcca atgcgcaaat tgatggacat caaatttcca aagctgaata tgaactggcc 180
cgccactcct tcagaccaag acttcaagcc acctatcgca acttgcacag gaaagacatc 240
gactgcgata gctcttgccc agcagactgg cctcccagtc ctctcgctcg atcgcgtcca 300
atgctgtcct caactatcaa ccggaagcgg gcgaccaaca gtggaagaac tgaaaggaac 360
gactcgtctg taccttgatg atcgcccttt ggtaaagggt atcattacag ccaagcaagc 420
tcatgaacgg ctcattgcgg aggtg 445