阅读说明：本技术 野葡萄VyPPR基因及其编码蛋白在干旱胁迫中的应用 (Wild grape VyPPR gene and application of encoding protein thereof in drought stress ) 是由 余义和 郭大龙 李旭飞 李敏 张贺程 张国海 于 2019-11-04 设计创作，主要内容包括：本发明公开了野葡萄VyPPR基因及其编码蛋白在干旱胁迫中的应用,核苷酸序列如SEQ:NO.1所示,编码序列全长为1874个核苷酸,开放阅读框为1590个核苷酸,该基因编码蛋白的氨基酸序列如SEQ:NO.2所示,可编码一个含529个氨基酸的蛋白,本发明通过利用强启动子驱动原理的转基因技术,将VyPPR基因的超量表达载体转入拟南芥中,从而获得转基因拟南芥植株。相对于转化空载体的拟南芥植株,超量表达VyPPR基因导致转基因拟南芥中抗逆相关物质的积累和抗旱相关基因的表达,葡萄克隆葡萄VyPPR基因,能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达,促进转基因植株抗旱性增强。(The invention discloses a wild grape VyPPR gene and application of a protein coded by the gene in drought stress, wherein a nucleotide sequence is shown as SEQ NO.1, the total length of a coding sequence is 1874 nucleotides, an open reading frame is 1590 nucleotides, an amino acid sequence of the gene coded protein is shown as SEQ NO.2, and the gene can code a protein containing 529 amino acids. Compared with an arabidopsis plant transformed with an empty vector, the VyPPR gene is overexpressed to cause accumulation of anti-stress related substances and expression of drought-resistant related genes in transgenic arabidopsis, and the grape VyPPR gene is cloned to increase accumulation of anti-stress related substances and expression of drought-resistant related genes in the transgenic plant and promote drought resistance enhancement of the transgenic plant.)

1. The wild grape VyPPR gene is characterized in that the nucleotide sequence is shown in SEQ NO.1, the total length of the coding sequence is 1874 nucleotides, and the open reading frame is 1590 nucleotides.

2. The vitis amurensis VyPPR gene of claim 1, wherein: the amino acid sequence of the gene coding protein is shown as SEQ No.2, and the gene can code a protein containing 529 amino acids.

3. The method for constructing the wild grape VyPPR gene plant overexpression vector according to any one of claims 1 or 2, wherein the method comprises the following steps: the method comprises the following steps:

(1) correctly inserting the ORF fragment of 1011 bp in total, which contains the VyPPR gene coding region, into a plant over-expression vector pCAMBIA 2300-GFP;

(2) according to the ORF sequence of the previously cloned VyPPR gene, adding enzyme cutting sites XbaI and KpnI at the 5' end of a primer VyPPR-ORF-F according to the enzyme cutting sites on a pCAMBIA2300-GFP vector,

GGGTCTAGAATGGCTCCTCCCCAAAATCAAC，

GGGGGTACCCTAGTACAGACTAATCAGACTC；

(3) and (2) taking the pMD18-T-VyPPR plasmid as a template, amplifying the plasmid by using VyPPR-ORF-XbaI-F and VyPPR-ORF-KpnI-R, recovering a target band, connecting the recovered target band to a pMD19-T cloning vector, and obtaining the plant expression vector pCAMBIA2300-VyPPR after connection, transformation, screening and verification.

4. The use of the wild grape VyPPR gene encoding protein of claim 1 or 2 in drought stress, wherein the gene is selected from the group consisting of: the specific method for overexpression of the grape VyPPR gene in Arabidopsis thaliana comprises the following steps:

(1) streaking agrobacterium containing recombinant plant expression vector on LB plate, and culturing in culture box;

(2) transferring the bacterial liquid into a centrifugal bottle or a centrifugal tube, centrifuging for 10min at the rotating speed of 4000 rpm, removing supernatant, collecting thalli, and resuspending in an osmotic buffer solution;

(3) soaking the pod-removed Arabidopsis thaliana flower in a penetrating fluid, removing redundant penetrating buffer solution on the Arabidopsis thaliana flower after soaking, putting the Arabidopsis thaliana flower in an incubator for continuous culture, carrying out normal management on the transformed Arabidopsis thaliana plant, and harvesting seeds when the pod is white;

(4) the VyPPR transgenic plants and wild-type plants obtained by primary screening of kanamycin are further identified at the DNA level, and total DNA is extracted by an improved SDS micro-extraction method.

5. The application of the wild grape VyPPR gene coding protein in drought stress according to claim 4, wherein the gene is selected from the group consisting of: and (3) identifying the drought resistance of the transgenic arabidopsis plants.

6. The application of the wild grape VyPPR gene coding protein in drought stress according to claim 4, wherein the gene is selected from the group consisting of: analyzing the physiological and biochemical characteristics of the transgenic arabidopsis plants, wherein the physiological and biochemical characteristic analysis comprises the determination of the water loss rate, the determination of the electrolyte leakage rate and the determination of the chlorophyll content.

Technical Field

The invention relates to the technical field of plant stress resistance gene identification and genetic engineering, in particular to application of wild grape VyPPR gene and coding protein thereof in drought stress.

Background

The PPR protein, also called a triangular five-membered repeat protein, is one of the largest protein families of terrestrial plants, having over 400 members in most species, and plays an important role in various stages of plant growth and development. THA8 found in maize, which contains only 4 PPR motifs, is involved in the splicing of intron type II of the transcript of the two genes ycf3 and trnA, and this protein, because it contains only a small amount of PPR motifs, does not bind to single-stranded RNA of either ycf3 or trnA, but is able to interact with the splicing factors WTF1 and RNC1, which are also involved in the splicing of the intron of trnRNAi. Fertility restorer protein RF5 in manglietia indica type rice, belongs to PLS family, has no ability of binding with target transcript atp6-orfH79, and interaction protein GRP162 can enter mitochondria along with the target transcript and is combined with the target transcript, meanwhile, RFC3 with WD40 structural domain participates in the construction of fertility restorer protein complex, and together with RF5, GRP162 and unknown protein, the complex is constructed into 400-500 kDa complex, and the complex mediates the cleavage of atp6-orfH79 transcript at nucleotide 1169 to inhibit the translation expression of male sterile protein ORFH 79. Another fertility restorer gene Rf6, which belongs to the P family PPR protein, comprises 20 PPR motifs, interacts with hexokinase HXK6, and researches show that RF6 can also form a protein complex with other proteins, the size of the protein complex is about 400-500 kDa, and RF6 does not interact with GRP162, which suggests that the protein complexes formed by RF6 and RF5 are different. The fertility restorer complex involved in RF6 also has a function of cleaving the atp6-orfH79 transcript, and cleaves at the 1238 th nucleotide of the transcript to inhibit accumulation of ORFH79 in mitochondria, thereby allowing normal development of male gametes into mature pollen with fertility.

The grapes are the second largest fruit in the world, have a long cultivation history, are various in variety, and have important edible value and economic value. In recent years, with the global climate change, drought events occur frequently around the world, and drought hazards also occur frequently in non-drought seasons or non-drought regions. Drought has serious influence on the growth and development process and yield quality of grapes, and becomes one of main factors for restricting the growth of grapes and improving the quality of fruits, and particularly, in recent years, the grape industry is greatly threatened due to global climate change and frequent occurrence of drought in the south of China. Under the large background of the problem of water shortage in the world, the exploration of drought-resistant grape resources and the research of drought-resistant genes of grapes have important scientific values and significance for improving the drought resistance of grapes, cultivating new drought-resistant varieties, saving water and cultivating and the like.

Disclosure of Invention

The invention aims to provide a cloned grape VyPPR gene which can increase the accumulation of anti-stress related substances and the expression of drought-resistant related genes in transgenic plants and promote the enhancement of the drought resistance of the transgenic plants.

In order to achieve the purpose, the invention provides the following technical scheme: the wild grape VyPPR gene and the application of the encoding protein thereof in drought stress have the nucleotide sequence shown in SEQ NO.1, the full length of the encoding sequence is 1874 nucleotides, and the open reading frame is 1590 nucleotides.

Preferably, the amino acid sequence of the protein encoded by the gene is shown in SEQ NO.2, and the gene can encode a protein containing 529 amino acids.

The method for constructing the wild grape VyPPR gene plant over-expression vector comprises the following steps:

(1) correctly inserting the ORF fragment of 1011 bp in total, which contains the VyPPR gene coding region, into a plant over-expression vector pCAMBIA 2300-GFP;

(2) according to the ORF sequence of the previously cloned VyPPR gene, adding enzyme cutting sites XbaI and KpnI at the 5' end of a primer VyPPR-ORF-F according to the enzyme cutting sites on a pCAMBIA2300-GFP vector,

GGGTCTAGAATGGCTCCTCCCCAAAATCAAC，

GGGGGTACCCTAGTACAGACTAATCAGACTC；

(3) and (2) taking the pMD18-T-VyPPR plasmid as a template, amplifying the plasmid by using VyPPR-ORF-XbaI-F and VyPPR-ORF-KpnI-R, recovering a target band, connecting the recovered target band to a pMD19-T cloning vector, and obtaining the plant expression vector pCAMBIA2300-VyPPR after connection, transformation, screening and verification.

The application of the wild grape VyPPR gene coding protein in drought stress, and the specific method for overexpression of the wild grape VyPPR gene in arabidopsis thaliana comprises the following steps:

(1) streaking agrobacterium containing recombinant plant expression vector on LB plate, and culturing in culture box;

(2) transferring the bacterial liquid into a centrifugal bottle or a centrifugal tube, centrifuging for 10min at the rotating speed of 4000 rpm, removing supernatant, collecting thalli, and resuspending in an osmotic buffer solution;

(3) soaking the pod-removed Arabidopsis thaliana flower in a penetrating fluid, removing redundant penetrating buffer solution on the Arabidopsis thaliana flower after soaking, putting the Arabidopsis thaliana flower in an incubator for continuous culture, carrying out normal management on the transformed Arabidopsis thaliana plant, and harvesting seeds when the pod is white;

(4) the VyPPR transgenic plants and wild-type plants obtained by primary screening of kanamycin are further identified at the DNA level, and total DNA is extracted by an improved SDS micro-extraction method.

Preferably, the drought resistance of transgenic arabidopsis plants is identified.

Preferably, the transgenic arabidopsis plants are analyzed for physiological and biochemical characteristics, including the determination of water loss rate, the determination of solute leakage rate and the determination of chlorophyll content.

The invention provides an application of wild grape VyPPR gene and its coded protein in drought stress, which has the following beneficial effects:

the invention transfers the overexpression vector of the VyPPR gene into arabidopsis thaliana by a transgenic technology utilizing a strong promoter (cauliflower mosaic virus 35S promoter) driving principle, thereby obtaining a transgenic arabidopsis thaliana plant. Compared with an arabidopsis plant transformed with an empty vector, the VyPPR gene is overexpressed to cause accumulation of anti-stress related substances and expression of drought-resistant related genes in the transgenic arabidopsis, and drought resistance of the transgenic plant is enhanced, so that the grape VyPPR gene is cloned, accumulation of anti-stress related substances and expression of drought-resistant related genes in the transgenic plant can be increased, and drought resistance enhancement of the transgenic plant is promoted.

Drawings

FIG. 1 shows the expression of the VyPPR gene of the present invention in different tissues of grape;

FIG. 2 shows the expression of the VyPPR gene of the present invention after low temperature treatment;

FIG. 3 is the expression of the VyPPR gene of the present invention after drought treatment;

FIG. 4 is the expression of the VyPPR gene of the present invention after salt stress;

FIG. 5 is the drought resistance identification of VyPPR transgenic Arabidopsis plants of the present invention;

FIG. 6 is a physiological characteristic analysis of VyPPR transgenic Arabidopsis plants of the present invention;

FIG. 7 shows the expression analysis of drought-resistant related genes in transgenic Arabidopsis plants of the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.