阅读说明：本技术 一个提高小麦籽粒淀粉合成的转录因子bZIP2及其应用 (Transcription factor bZIP2 for improving starch synthesis of wheat grains and application thereof ) 是由 张爱民 刘冬成 宋艳红 罗光彬 孙家柱 李欣 阳文龙 于 2019-09-04 设计创作，主要内容包括：本发明公开了一个能提高小麦籽粒淀粉合成的转录因子<I>bZIP2</I>及其应用,该转录因子是<I>TubZIP2</I>基因,以及<I>TubZIP2</I>基因在普通小麦中同源基因<I>TabZIP2</I>的三个拷贝：<I>TabZIP-A2</I>、<I>TabZIP-</I><I>B2</I>和<I>TabZIP-D2</I>,其编码区是分别由SEQ ID No.1、No.2、No.3和No.4所示核苷酸序列组成。在<I>TubZIP2</I>的过表达小麦株系中,<I>AGPL-cyto、AGPS-</I><I>cyto</I>和<I>SBEIIa</I>的表达在胚乳灌浆的中后期较野生型的显著提高,而其它淀粉合成相关基因(SSRG)的表达量没有变化,粒长和千粒重也明显增加。敲除普通小麦中的基因<I>TabZIP2</I>,敲除系的总淀粉和支链淀粉含量显著降低,粒长和千粒重也显著减小。<I>TubZIP2</I>过表达系和<I>TabZIP2</I>敲除系的株高、有效分蘖、小穗数和粒长没有明显变化。(The invention discloses a transcription factor capable of improving the starch synthesis of wheat grains bZIP2 And uses thereof, the transcription factor is TubZIP2 A gene, and TubZIP2 gene homology gene in common wheat TabZIP2 Three copies of (a): TabZIP‑A2 、 TabZIP‑ B2 and TabZIP‑D2 the coding regions are respectively composed of SAnd the nucleotide sequences shown in EQ ID No.1, No.2, No.3 and No. 4. In that TubZIP2 In the case of the over-expressed wheat line of (1), AGPL‑cyto、AGPS‑ cyto and SBEIIa the expression of the protein is obviously improved in the middle and later periods of endosperm filling compared with the wild type, while the expression quantity of other Starch Synthesis Related Genes (SSRG) is not changed, and the grain length and thousand kernel weight are also obviously increased. Knocking out genes in common wheat TabZIP2 The total starch and amylopectin content of the knockout line is obviously reduced, and the grain length and thousand grain weight are also obviously reduced. TubZIP2 Over-expression lines and TabZIP2 the plant height, effective tillering, small spike number and grain length of the knockout line have no obvious change.)

1. The transcription factor for improving the synthesis of the starch of the kernel in the Wularg wheat is characterized in that: the transcription factor isTubZIP2A gene or isTubZIP2Gene homology gene in common wheatTabZIP2Three copies of (a):TabZIP-A2、TabZIP-B2andTabZIP-D2saidTubZIP2Gene, gene,TabZIP-A2Gene, gene,TabZIP-B2Gene, gene,TabZIP-D2The coding regions of the genes are respectively SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.2The nucleotide sequence shown in ID No. 4.

2. A recombinant vector comprising the transcription factor of claim 1.

3. An expression vector, preferably a plant expression vector, comprising the transcription factor of claim 1.

4. Use of the transcription factor of claim 1 to increase expression of the SSRG gene in the grain filling stage in wheat.

5. The use of claim 4, wherein: transforming the transcription factor into wheat, and screening a positive transgenic line over expressing the transcription factor.

6. A method of overexpressing the transcription factor of claim 1 in endosperm, wherein: and (3) transforming the endosperm of the common wheat by using the overexpression vector containing the transcription factor, preferably transforming the endosperm 15 days after the common wheat blossoms by using a gene gun method.

7. A primer set for amplifying the gene of claim 1, wherein: the sequence of the forward primer of the primer pair is shown as SEQ ID No.5, and the sequence of the reverse primer is marked by SEQ ID No. 6.

8. Use of the transcription factor of claim 1 for increasing the expression of the SSRG gene and promoting starch accumulation in wheat, characterized in that: and (3) converting the over-expression vector of the transcription factor into common wheat, and screening to obtain a positive transgenic line with increased starch content.

9. Use according to claim 8, characterized in that: the transcription factor isTubZIP2。

10. The transcription factor of claim 1 for reducing SSRG gene expression in Triticum aestivumUse of starch accumulation, characterized in that: gene knockout by CRISPR/Cas9 technologyTabZIP2Three copies of (a):TabZIP-A2、TabZIP-B2orTabZIP-D2And screening out positive transgenic lines.

Technical Field

The invention relates to the field of genetic engineering, in particular to a transcription factor for improving the expression of wheat starch synthesis related genes and application thereof.

Background

Wheat, one of the major crops in the world, not only provides protein, vitamins and dietary fiber for human health, but also is a major source of starch and energy in the human diet. Starch is the main storage component in the endosperm of wheat grains, accounting for 60-70% of the total weight of grains and 70-85% of the total weight of flour.

Starch synthesis in wheat endosperm cells involves a complex series of reactions requiring the involvement of a series of enzymes. At the start of starch synthesis, AGPase catalyzes the conversion of glucose-1-phosphate (G1 c-1-P) and Adenosine Triphosphate (ATP) to adenosine diphosphate glucose (ADP-glucose), a glucose donor for amylose and amylopectin synthesis. Thereafter, granule-bound starch synthase (GBSS) catalyzes the ADP-glucose to finally form amylose, while Starch Synthase (SS), Starch Branching Enzyme (SBE) and debranching enzyme (DBE) catalyze the ADP-glucose transfer to highly branched glucan branches to finally form amylopectin. The content of the starch of the grains can be effectively improved by regulating and controlling the expression of the starch synthesis related gene, thereby improving the yield of wheat. However, to date, there have been few reports on transcriptional regulation of starch synthesis in cereal grains. WhereinRSR1AndOsbZIP58is in riceIdentifying the obtained starch synthesis regulating factor;ZmNAC36、ZmbZIP91、ZmEREB156andSUSIBA2are transcription factors that regulate starch synthesis identified in maize and barley, respectively. In wheat, except for riceRSR1Homologous geneTaRSR1There is no report on the systematic study of starch synthesis regulation mechanism.

Disclosure of Invention

The invention identifies a transcription factor Co-expressed with Starch Synthesis Related Genes (SSRG) in endosperm filling period by Weighted Gene Co-expression Network Analysis (WGCNA)TubZIP2。TubZIP2And homologous gene thereof in common wheatTabZIP2Can be remarkably improvedAGPL-cyto、AGPS-cytoAndSBEIIathe expression of the gene further improves the total starch and amylopectin content of the wheat grains. At the same time, the user can select the desired position,TubZIP2andTabZIP2has no any adverse effect on other important agronomic traits of wheat.

The invention is helpful for people to understand the regulation and control of wheat starch synthesis,TubZIP2andTabZIP2can also be used as an important target gene for genetic engineering and traditional breeding to culture a new wheat variety with high yield and high quality.

The inventor identifies and obtains the genome data analysis of endosperm of the grain filling stage of the Wulare diagram wheat G1812TubZIP2Has similar expression pattern with partial SSRG gene. The TubZIP2 subcellular location is in the nucleus, and 1-128aa has transcriptional activation activity. In the dual-luciferase reporter gene system analysis and the wheat endosperm transient overexpression analysis,TubZIP2promotion ofAGPL-cyto、AGPS-cytoAndSBEIIaactivity of a gene promoter. In thatTubZIP2In the case of the over-expressed wheat line of (1),AGPL-cyto、AGPS-cytoandSBEIIathe expression of (a) is obviously improved in the middle and later stages of endosperm filling compared with the wild type, and the expression quantity of other SSRG genes is not changed. Correspondingly, the total starch content and the amylopectin content of the overexpression line are obviously improved, and the grain length and the thousand grain weight of the overexpression line are also obviously increased. In contrast, knockouts using CRISPR/Cas9 technologyTubZIP2In the generalHomologous genes in wheatTabZIP2The total starch content and amylopectin content of the knockout line are obviously reduced, and the grain length and thousand grain weight are also obviously reduced.TubZIP2Over-expression lines andTabZIP2the plant height, effective tillering, small spike number and grain length of the knockout line have no obvious change. In any case, it is preferable that,TubZIP2andTabZIP2promotes the synthesis of the starch of the wheat grains, and has no negative influence on other important agronomic traits.

The invention aims to provide a transcription factor for regulating and controlling the expression of an SSRG geneTubZIP2The transcription factor promotes starch synthesis.

The transcription factor provided by the inventionTubZIP2Is derived from wheat with a Wulare diagram and has a coding region consisting of a nucleotide sequence shown as SEQ ID No. 1. The DNA shown in SEQ ID No.1 has a length of 1722 bp (excluding stop codon), belongs to the bZIP family, and encodes a protein of 574 amino acid residues.TubZIP2The homologous gene in common wheat isTabZIP2It has three copiesTabZIP-A2、TabZIP-B2AndTabZIP-D2the coding regions are respectively nucleotide sequences shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, the length of the coding regions is 1722 bp, and 574 protein with amino acid residues is coded.

Meanwhile, the present invention provides a recombinant vector comprising the transcription factor of claim 1.

The invention also provides an expression vector containing the transcription factor, preferably a plant expression vector.

The invention provides an application of the transcription factor in improving the expression of SSRG genes in the filling stage in wheat; the application is to transform the transcription factor into wheat and screen a positive transgenic line over expressing the transcription factor.

The invention also provides a method for over-expressing the transcription factor in endosperm, which is to transform the endosperm of common wheat by using an over-expression vector containing the transcription factor, preferably to transform the endosperm 15 days after the common wheat blossoms by using a gene gun method.

Also, the present invention provides amplificationTubZIP2AndTabZIP2full length of coding regionThe primer pair is used, wherein the sequence of the forward primer is shown as SEQ ID No.5, and the sequence of the reverse primer is shown as SEQ ID No. 6.

The invention also provides a method for determining and regulating the candidate transcription factor expressed by the SSRG gene by utilizing co-expression analysis, which finds the transcription factor with a similar expression mode to the SSRG gene by analyzing the transcriptome of the endosperm of the wheat in the filling stage of the Ural chart through RNA-Seq.

The invention provides a method for detecting the regulation intensity of a transcription factor on an SSRG gene promoter region, which comprises the steps of co-transforming an overexpression vector of the transcription factor and a report vector of the SSRG gene promoter driving the expression of Firefly luciferase (Firefly luciferase) gene into a wheat protoplast cell, and detecting the intensity of the activity of the transcription factor regulating the SSRG gene promoter region by taking Renilla luciferase (Renilla luciferase) as an internal reference.

The invention provides a method for detecting the regulation and control of SSRG gene expression by transcription factor through endosperm overexpression, which comprises the steps of converting an overexpression vector of the transcription factor into endosperm 15 days after common wheat blossoms by a gene gun method, carrying out dark culture in a hypertonic culture medium for 48 hours, and detecting the change of SSR gene expression in the endosperm by RT-PCR.

The invention provides a Ular pattern wheatTubZIP2Can be used for up-regulating immature seeds of common wheatAGPL-cyto、AGPS-cytoAndSBEIIathe expression of the gene and finally the content of the total starch and the amylopectin of the mature seeds are improved. Its homologous gene in common wheatTabZIP2Has the same function. Therefore, the temperature of the molten metal is controlled,bZIP2has important significance for improving the starch quality of wheat.

Drawings

FIG. 1 shows a schematic view of aTubZIP2Evolutionary tree analysis, subcellular localization, transcriptional activation activity and expression pattern analysis. Wherein, A is the structure of TubZIP2, wherein 128-179 amino acids (aa) represent the core region of bZIP family transcription factor; b is an evolutionary tree analysis displayTubZIP2And homologous genes thereof are conserved in wheat families, and have a far relation with homologous genes from other families of the gramineae family; c is the N-terminal (1-127 aa) of TubZIP2 with transcriptional activation activity; d is TubZIP2 located in cell nucleus;e isTubZIP2In thatT. urartuPerforming specific expression in mature endosperm; f isTubZIP2And a part of the SSRG gene inT. urartuCo-expression in the grouting process.

FIG. 2TubZIP2In vitro enhancementAGPL-cyto、AGPS-cytoAndSBEIIatranscription of the gene. A is a schematic diagram of a report vector, an expression vector and an internal reference in a dual-luciferase reporter gene system, wherein REN: renilla luciferase; LUC: firefly luciferase; ubi: ubiquitin; p: a promoter; ter: and (6) terminating. B isTubZIP2Significant improvement in dual luciferase reporter systemsAGPL-cyto、AGPS-cytoAndSBEIIapromoter activity of the gene; c isTubZIP2A schematic representation of the overexpression of the vector in the endosperm; d isTubZIP2Increased overexpression in endospermAGPL-cyto、AGPS-cytoAndSBEIIatranscription of the gene.

FIG. 3TubZIP2Increasing wheat over-expression in linesAGPL-cyto、AGPS-cytoAndSBEIIathe level of transcription of the gene. A and B represent the results of RNA-seq and RT-PCR, respectivelyTubZIP2Overexpression in the immature endosperm of transgenic wheat; C. e and G are in endosperm 7 days, 14 days and 21 days after anthesis in RNA-seq analysis, respectivelyAGPL-cyto、AGPS-cytoAndSBEIIaa change in the level of transcription; D. f and H represent endosperm 7 days, 14 days and 21 days after RT-PCR validationAGPL- cyto、AGPS-cytoAndSBEIIachange in the amount of gene expression.

FIG. 4TubZIP2The total starch content and the amylopectin content of the overexpression system are improved. A isTubZIP2Remarkably improving the total starch content of the over-expression line planted in the 2017-plus 2018 Beijing, the dike and the Zhao county; b, finding that the number of A-type starch granules in the over-expression line seeds planted in Zhao county is more than that of wild type grains in scanning comparison by an electron microscope, wherein A and B respectively represent A-type and B-type starch granules; C. d and E are the amylopectin content, the amylose content and the ratio of amylose to amylopectin of the three-site overexpression lines, respectively; f is the higher peak viscosity of the overexpression line of Beijing in RVA analysis compared with the wild type.

FIG. 5TubZIP2Increasing grain of overexpression lineGrain weight and grain width. A and B are respectively wild type and overexpression line plant and spike morphology; C. d and E are respectively the plant height, effective tillering and the number of spikelets per spike of the wild type and the overexpression line; f is the grain length and grain width shape of wild type and over-expression line grains; g isTubZIP2No influence on the grain length of the transgenic line; h and I are eachTubZIP2The grain width and thousand grain weight of the over-expression line are improved.

FIG. 6TabZIP2In vitro enhancementAGPL-cyto、AGPS-cytoAndSBEIIatranscription of the gene. A isTabZIP2Specific expression in common wheat endosperm; b isTabZIP2AndAGPL-cyto、AGPS-cytoandSBEIIaco-expression; c isTabZIP2A schematic representation of the overexpression of the vector in the endosperm; d isTabZIP2Increased overexpression in endospermAGPL-cyto、AGPS- cytoAndSBEIIatranscription of the gene.

FIG. 7TabZIP2The total starch content and amylopectin content of the knockout line were reduced. A is Beijing, on the dike and Zhao countyTabZIP2The total starch content of the knockout system is obviously reduced; b, finding that the number of A-type starch grains in the knock-out seeds planted in Zhao county is less than that of wild type grains in electron microscope scanning comparison, wherein A and B respectively represent A-type and B-type starch grains; c is the amylopectin content of the three-site knockout line; d is the lower peak viscosity of the Beijing-planted knockout line compared to the wild type in the Rapid Viscometry (RVA) analysis; e and F show a significant reduction in grain width and thousand grain weight, respectively, for the knockout line.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.