阅读说明：本技术 利用油菜BnaA6YUC6基因改良油菜分枝角度 (Rape BnaA6YUC6 gene for improving rape branching angle ) 是由 胡琼 郝梦宇 成洪涛 丁炳莉 王文祥 刘佳 梅德圣 付丽 于 2019-10-15 设计创作，主要内容包括：本发明属于植物基因工程技术领域,具体涉及利用油菜BnaA6YUC6基因改良油菜分枝角度。本发明利用过表达油菜BnaA06YUC6基因的载体转化油菜,用以改良油菜的分枝角度,其中,所述油菜BnaA06YUC6基因的核苷酸序列如序列表SEQ ID NO：1所示；其蛋白质序列如序列表SEQ ID NO:2所示。以甘蓝型油菜品种中双6号为受体,构建过表达油菜BnaA06YUC6基因的转基因植株。功能验证表明,油菜BnaA06YUC6基因所表现出来的这种功能可以用来改良农作物的分枝结构,如减小分枝角度等性状。(The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for improving rape branch angle by utilizing rape BnaA6YUC6 gene. The invention utilizes a vector for over-expressing rape BnaA06YUC6 gene to transform rape so as to improve the branching angle of rape, wherein the nucleotide sequence of the rape BnaA06YUC6 gene is shown as a sequence table SEQ ID NO:1 is shown in the specification; the protein sequence is shown in a sequence table SEQ ID NO. 2. A transgenic plant of over-expression rape BnaA06YUC6 gene is constructed by taking double No. 6 in cabbage type rape variety as a receptor. Functional verification shows that the function expressed by the rape BnaA06YUC6 gene can be used for improving the branch structure of crops, such as reducing the characters of branch angle and the like.)

The application of the BnaA06YUC6 gene in improving the branching angle of rape is characterized in that the nucleotide sequence of the BnaA06YUC6 gene is shown as SEQ ID NO:1 is shown.

The application of the BnaA06YUC6 gene in improving the branching angle of rape, which is characterized in that the protein sequence coded by the BnaA06YUC6 gene is shown as SEQ ID NO:2, respectively.

3. An expression vector pBI121S-BnaA06YUC6 for transforming the branching angle of Brassica napus is characterized by comprising the nucleotide sequence shown in a sequence table SEQ ID NO:1, and by taking pBI121S as an expression vector, the nucleotide sequence shown in SEQ ID NO:1 is obtained by inserting BnaA06YUC6 gene into PBI121S vector 35S constitutive promoter downstream.

4. The use of the BnaA06YUC6 gene as claimed in claim 1, wherein said brassica napus is transformed by overexpressing said BnaA06YUC6 gene.

5. The use of the BnaA06YUC6 gene as claimed in claim 2, wherein said brassica napus is transformed by overexpressing said BnaA06YUC6 gene.

Technical Field

The invention belongs to the technical field of plant transgenosis, and particularly relates to a method for improving a rape branch angle by utilizing a rape BnaA06YUC6 gene.

Background

Rape is the first major oil crop in China, and the planting area and the yield are in the forefront of the world. The oil yield of the rape accounts for more than 55 percent of the total yield of the whole domestic oil crops, and the rape occupies a main position in the domestic vegetable oil supply.

The ideal plant type is a key factor for improving the crop yield. The breeding of the ideal plant type material with both form and function breaks the situation that the unit yield of the crops is not in wandering state is one of the main targets of breeding. In the cultivation of monocotyledonous crops (such as rice, corn, wheat and the like), the small inclination angle and the erect leaf are important parameters of plant canopy, so that the whole plant can be irradiated by sunlight from top to bottom, the photosynthetic efficiency is improved, and the planting density is increased (Murchie et al, 2009; Zhu et al, 2010; Murchie and Reynolds, 2012; Drewry et al, 2014; Mansfield and Mumm, 2014). With the popularization of the direct seeding technology of rape, the improvement of the planting density through plant type improvement has important significance for improving the yield and implementing mechanized harvesting. Compact rape has erect leaves in seedling stage and small included angle of branches in adult stage, so that a single plant occupies less space and is suitable for high-density planting, the improvement of the light energy utilization rate of a group is facilitated, the biological yield is increased, the economic coefficient is improved, and the higher yield (Cai et al, 2016) is obtained. Meanwhile, the branch parts of the compact rape are properly improved under the condition of close planting, the effective branch number and the branch length are partially reduced, the maturity period of the rape is relatively concentrated, plants are not crossed with each other, the mechanical harvesting of the rape is more favorably carried out, and the loss caused by machine harvesting is reduced. Therefore, the rape plant type is improved, the rape branch angle is reduced, the rape yield can be improved, the implementation of mechanical production can be promoted, and the long-term goal of rape production development in China is met.

YUCCA plays an important role in the auxin synthesis pathway. The YUCCA protein catalyzes the conversion of indole-3-pyruvate to indole-3-acetic acid, completing the final synthesis of auxin. It was found by the studies of overexpression and loss-of-function multi-mutants that abnormal expression of the YUCCA gene resulted in defects in plant growth and development, which were associated with excessive synthesis or deletion of auxin (Cheng et al, 2006). Over-expression or suppression of YUCCA gene expression in rice, plants show auxin-over and auxin-insensitive phenotypes, respectively (Yamamoto et al, 2007). Overexpression of the YUCCA6 gene in arabidopsis enhances the apical dominance of the plants and delays the senescence process (Cha et al, 2015). Researchers over-express the YUCCA6 gene in potato and tobacco, plants also showed apical dominance, and IAA content and auxin-related gene expression were significantly increased (Kim et al, 2013; Ke et al, 2015). At present, no report related to regulation of plant types or branch angles of plants by using a YUCCA gene is seen at home and abroad.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and reduce the branching angle of rape by using a rape branching angle regulating gene BnaA06YUC 6. The invention provides a nucleotide sequence of BnaA06YUC6 gene and a protein sequence coded by the same. In the embodiment, the vector of the over-expression rape BnaA06YUC6 gene is used for transforming the double 6 in the cabbage type rape variety, so that the double 6 in the cabbage type rape variety shows the characteristics of reduced branching angle and compact plant type.

In the early stage, a candidate QTL interval for controlling the branching angle on an A06 chromosome is obtained by a QTL-seq method, a differential SNP locus is selected in the candidate QTL interval to obtain the differential SNP locus capable of causing amino acid missense mutation of a BnaA06YUC6 gene, and then a branching angle regulation gene BnaA06YUC6 is screened, and further the BnaA06YUC6 gene is determined to reduce the branching angle of No. two 6 in the semi-winter cabbage type rape by a genetic transformation method. The invention identifies a candidate gene BnaA06YUC6 for controlling the branching angle by a QTL-seq method for the first time, and reduces the branching angle of rape plants by over-expressing the gene, so that the plant types of the rape plants become more compact. Through the literature search, no research report about the regulation of rape branch angle by using cabbage type rape YUCCA gene is found in China, and no related patent application publication or use is found.

The invention also includes: the separation and cloning of rape BnaA06YUC6 gene, transgenic function research and application, etc.

The technical scheme of the invention is realized by the following method:

1. rape BnaA06YUC6 gene source and protein structure analysis

The previous work of the invention is to construct an F2 segregating population by using Huyou oil 19 (a known rape variety) which is a rape variety with large branching angle and Purler (a rape variety from Australia) which is a rape variety with small branching angle (the method for constructing the F2 segregating population is a conventional method), and to obtain a candidate QTL interval for controlling the branching angle on A06 chromosome by selecting extreme single plants and using a DNA mixed pool high-throughput sequencing technology (QTL-seq). And selecting SNP sites in the region screened under the 95% confidence interval in a 1M window in the candidate QTL interval, and annotating the SNP sites. A differential SNP locus is obtained in the interval, and can cause amino acid missense mutation of BnaA06YUC6 gene on rape A06 chromosome (Wang et al, 2016).

The full-length coding sequence of BnaA06YUC6 was obtained by PCR amplification. Primers are designed according to CDS sequence information of BnaA06YUC6 gene predicted by the Damor genome, and the full-length CDS sequence of BnaA06YUC6 is amplified. Wherein: the primer sequence for amplifying the full-length coding sequence of BnaA06YUC6 is as follows: (forward primer BnaA06YUC6F1: ATGGATTGGAAGAAAGAGATG; reverse primer BnaA06YUC6R1: TTAGGCTTGATCAGGTTTACT). The cDNA sequence of the cabbage type rape Purler is cloned to a rape BnaA06YUC6 gene, and the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO:1, the protein sequence coded by the gene is shown in a sequence table SEQ ID NO:2, the total length of the coding sequence is 1299bp, and 432 amino acids are coded.

The protein coded by the rape BnaA06YUC6 gene has a typical FMO-like structural domain, and the specific structure is shown in figure 1.

2. Construction of overexpression rape BnaA06YUC6 gene vector

The nucleotide sequence (SEQ ID NO: 1) and the protein sequence (SEQ ID NO: 2) of the rape BnaA06YUC6 gene were used in the examples of the present invention. The expression vector for over-expressing rape BnaA06YUC6 gene is obtained by taking a pBI121S vector as a basic vector for expression and inserting the rape BnaA06YUC6 gene sequence into the downstream of a 35S constitutive promoter of the pBI121S vector by utilizing a restriction enzyme site BamH I. The structure of the expression vector is shown in FIG. 2.

3. Arabidopsis thaliana transformed by rape BnaA06YUC6 gene and DNA detection

The invention uses wild arabidopsis thaliana variety Columbia as receptor, utilizes agrobacteria mediated inflorescence dip-dyeing method to introduce rape BnaA06YUC6 gene into inflorescence of wild arabidopsis thaliana variety Columbia through overexpression vector, and uses kanamycin resistance (conventional method) to screen transgene T₁Generating seeds to obtain transgenic arabidopsis thaliana plants over-expressing rape BnaA06YUC6 gene. And (3) taking leaves at the seedling stage and extracting genome DNA by using a conventional CTAB method. PCR amplification is carried out by using a labeled primer NPTII (a forward primer NPTIIF: GATGGATTGCACGCAGGT; a reverse primer NPTIIR: TCGTCAAGAAGGCGATAGA) on the transformation vector (the PCR amplification system is that DNA1 mul, 2 XPCR mix 10 mul, primers F and R are 0.5 mul respectively, double distilled water is 8 mul, the total volume is 20 mul, the PCR reaction program is that 95 ℃ is 5min, 95 ℃ is 30s, 57 ℃ is 30s, 72 ℃ is 40s, 30 cycles, 72 ℃ is 5min, and 25 ℃ is 1min), the positivity of the transformation material is detected, and finally a positive plant is obtained.

4. Transformation test and DNA detection of rape BnaA06YUC6 gene

The invention takes double No. 6 (a known rape variety, oil crop research institute of Chinese academy of agricultural sciences) in a cabbage type rape variety as a transgenic receptor, introduces a rape BnaA06YUC6 gene overexpression vector (as described above) into the double No. 6 cut short hypocotyls in the cabbage type rape variety by utilizing an agrobacterium-mediated infection method, and screens positive plants by kanamycin resistance to obtain a transgenic plant overexpressing the rape BnaA06YUC6 gene. The double No. 6 rape variety transformed by rape BnaA06YUC6 gene is transplanted to a growing room (artificial culture room), and the leaf is taken at seedling stage to extract genome DNA by conventional CTAB method. The PCR amplification (PCR amplification method is the same as above) is carried out by using the marker primer NPTII (forward primer NPTIIF: GATGGATTGCACGCAGGT; reverse primer NPTIIR: TCGTCAAGAAGGCGATAGA) in the transformation vector, the positive of the transformation material is detected, and finally, a positive plant is obtained.

5. Expression quantity detection of transgenic BnaA06YUC6 gene Arabidopsis thaliana plant

The RNA of young single leaf of an arabidopsis transformed plant is extracted by adopting a universal Trizol method (Trizol reagent used in the method is purchased from Dalian technologies GmbH Co., Ltd.) and is subjected to reverse transcription, then an arabidopsis AtActin gene (login number AT5G09810.1) is used as an internal reference, amplification primers are (a forward primer AtActinF: GGTTCGTGGTGGTGAGTTTG and a reverse primer AtActinR: GTATCGGGTGACAATGCAGC), a specific amplification primer of a BnaA06YUC6 gene is designed (a forward primer BnaA06YUC6F2: ATGGATTGGAAGAAAGAGATG and a reverse primer BnaA06YUC6R2: TTAGGCTTGATCAGGTTTACT) to amplify a partial segment of the BnaA06YUC6 gene of rape (a PCR amplification system is DNA1 mul, 2 XPCR mix 10 mul, each of the primers F and R is 0.5 mul, double distilled water UC 8 mul, the total volume is 20 mul, the PCR reaction program is 95 ℃ 5min, 95 ℃ 30s, 57 ℃ 30s, 40 ℃ and 829 72 min, and the cycle change of the gene expression quantity is 4 ℃ of the BnaA 06. multidot. 95). The expression level of the gene in BnaA06YUC6 transgenic plant is obviously increased compared with the expression level of a control arabidopsis wild type Columbia plant. The results are shown in FIG. 3.

6. Detection of expression level of rape transformed with BnaA06YUC6 gene

Extracting RNA of young individual leaves of rape by using a universal Trizol method (the method is the same as the method), carrying out reverse transcription, and amplifying a partial fragment of rape BnaA06YUC6 gene by using a specific amplification primer of BnaA06YUC6 gene (a forward primer BnaA06YUC6F3: CTTGTCTCAAAAAGAAAGGA; a reverse primer BnaA06YUC6R3: AATCAAGAGGAAACGGTCAA); rape BnaActin gene (BnaC02g00690D) is used as an internal reference, the amplification primer is (forward primer BnaActinF: TCTGGCATCACACTTTCTACAACGAGC; reverse primer BnaActinR: CAGGGAACATGGTCGAACCACC), and the change condition of BnaA06YUC6 gene expression level is analyzed (the PCR amplification method is the same as the method above). The expression level in BnaA06YUC6 transgenic plant is obviously increased compared with the expression level in the double No. 6 plant in the control cabbage type rape variety. The results are shown in FIG. 4.

7. Investigation and statistics of branch angle characters of rape BnaA06YUC6 gene transformed Arabidopsis thaliana plants

Two families of BnaA06YUC6 transgenic Arabidopsis positive plants with significantly higher expression level than wild type control Arabidopsis wild type Columbia harvest seeds, plant the next generation, take leaves at seedling stage and extract genome DNA by conventional CTAB method. The positive test was carried out by PCR amplification using the labeled primer NPTII (forward primer NPTIIF: GATGGATTGCACGCAGGT; reverse primer NPTIIR: TCGTCAAGAAGGCGATAGA) on the transformation vector. And measuring the included angle between each branch and the main branch of the positive single plant from the bottom to the top by using a protractor, and taking the average value as the branch angle of the single plant. The branching angle in the BnaA06YUC6 transgenic positive plant is significantly reduced compared with the branching angle of the control Arabidopsis wild type Columbia. The specific results are shown in FIG. 5.

8. Investigation and statistics of rape BnaA06YUC6 gene transformed rape plant branching angle character

Two families with obviously increased expression quantity compared with double No. 6 in the contrast cabbage type rape in BnaA06YUC6 cabbage type rape positive plants are harvested for seeds, the next generation is planted, and leaves are taken at the seedling stage to extract genome DNA by using a conventional CTAB method. The positive test was carried out by PCR amplification using the labeled primer NPTII (forward primer NPTIIF: GATGGATTGCACGCAGGT; reverse primer NPTIIR: TCGTCAAGAAGGCGATAGA) on the transformation vector. And measuring the included angle between each branch and the main branch of the positive single plant from the bottom to the top by using a protractor, and taking the average value as the branch angle of the single plant. The branching angle in BnaA06YUC6 transgenic positive plants is obviously smaller than the branching angle of double No. 6 in control brassica napus. The specific results are shown in FIG. 6.

9. Determination of enzymatic Activity of rape BnaA06YUC6 protein

In this example, the nucleotide sequence (shown in SEQ ID NO: 1) and the protein sequence (shown in SEQ ID NO: 2) of the rape BnaA06YUC6 gene were used. The prokaryotic expression vector structure diagram of the BnaA06YUC6 gene is obtained by taking a prokaryotic expression vector pCold-TF (a common commercial prokaryotic expression vector purchased from Takara company) as a basic vector and inserting the full-length CDS sequence of the BnaA06YUC6 gene into the downstream of a CSPA promoter and a His tag of the pCold-TF vector by utilizing restriction enzyme sites BamH I and EcoR I, wherein the structural diagram of the expression vector is shown in figure 7. The constructed prokaryotic expression vector is introduced into an escherichia coli strain BL21 by a conventional heat shock method, and IPTG is added for induction expression. After the BnaA06YUC6 protein is determined to be capable of inducing expression, the protein is purified by using a His tag by using a general affinity chromatography method, and the empty carrier tag protein is induced to express and purified.

To 1 XPBS buffer (available from Solebao, pH7.4) was added the purified protein solution, together with FAD (flavin adenine dinucleotide) at a final concentration of 40. mu.M, IPA (indole-3-pyruvate) at 100. mu.M and NADPH (reduced nicotinamide adenine dinucleotide phosphate) at 1mM, and the reaction was carried out in a water bath at 30 ℃ for 30 minutes, after which the reaction was terminated with 1/10 volumes of HCl (1N). And (3) measuring the content of each component by using a liquid chromatography-mass spectrometer together with the reacted sample and IPA and IAA (indole-3-acetic acid) standard substances. In the reaction system containing the BnaA06YUC6 protein, a clear IAA product peak can be detected, while the reaction system added with the control His tag protein does not generate a clear IAA peak, which indicates that the BnaA06YUC6 protein cloned by the invention can convert precursor IPA into IAA. The results are shown in FIG. 8.

Drawings

FIG. 1: structural map of rape BnaA06YUC6 gene.

FIG. 2: an expression vector structural map of an overexpression rape BnaA06YUC6 gene.

FIG. 3: and (3) a semi-quantitative RT-PCR detection result of the transgenic arabidopsis thaliana with the rape BnaA06YUC6 gene.

FIG. 4: semi-quantitative RT-PCR detection result of rape BnaA06YUC6 transgenic plant.

FIG. 5: BnaA06YUC6 transformed Arabidopsis thaliana plant T₂The survey results of the branch angle of the family.

FIG. 6: rape BnaA06YUC6 transgenic plant T₀Instead of the branch angle measurement results.

FIG. 7: a prokaryotic expression vector structural map of rape BnaA06YUC6 gene expressed in vitro.

FIG. 8: the result of enzyme activity determination of rape BnaA06YUC6 protein expressed in vitro. Description of reference numerals: FIG. 8, panel A shows the elution peaks for IPA and IAA standards (where a represents IPA and b represents IAA); FIG. 8B shows that after the addition of empty carrier-tagged protein to the reaction system, substrate IPA but no IAA was detected; FIG. 8C shows that after BnaA06YUC6 protein was added to the reaction system, both the substrate IPA and the product IAA could be detected.

Detailed Description

Description of sequence listing:

SEQ ID NO. 1 of the sequence Listing is the nucleotide sequence of the BnaA06YUC6 gene (wherein 1-1296bp is the coding region of the gene).

The sequence table SEQ ID NO 2 is a protein sequence coded by BnaA06YUC6 gene.