阅读说明：本技术 提高豆科植物脂肪酸含量在促进其根瘤形成中的应用 (Application of increasing content of fatty acid in leguminous plants in promoting root nodule formation ) 是由 胡赞民 范成明 陈宇红 郭雪洁 郭徐鹏 赵敬会 陆明洋 傅永福 王晓波 于 2019-08-09 设计创作，主要内容包括：本发明提供了提高豆科植物脂肪酸含量在促进其根瘤形成中的应用。本发明将来源于椭圆小球藻的CeDGAT1和CectGPDH2基因利用遗传转化的方法获得大豆转基因材料,研究发现所获得的转基因大豆在表型上未出现不良的农艺性状；同时发现CeDGAT1和CectGPDH2的转基因株系,其百粒重和种子中的总脂肪酸均提高；并且根瘤数提高40％以上；氮利用率显著提高。说明提高大豆脂肪酸含量,能够促进豆科植物的根瘤形成,进而提高豆科植物的氮利用率,实现豆科植物的增产,本发明这一发现可用于豆科植物的增产增收。因此我们的发明结果可用于通过基因工程或非基因工程提高豆科植物脂肪酸含量来促进豆科植物根瘤形成。(The invention provides an application of increasing the content of fatty acid in leguminous plants in promoting the formation of nodules of the leguminous plants. The invention obtains soybean transgenic material by utilizing a genetic transformation method for CeDGAT1 and CectGPDH2 genes derived from Chlorella ellipsoidea, and researches show that the obtained transgenic soybean does not have undesirable agronomic characters on phenotype; meanwhile, transgenic lines of CeDGAT1 and CectGPDH2 are found, and the hundred grain weight and the total fatty acid in seeds are increased; the number of the root nodules is increased by more than 40 percent; the nitrogen utilization rate is obviously improved. The invention shows that the soybean fatty acid content is increased, the nodule formation of leguminous plants can be promoted, the nitrogen utilization rate of the leguminous plants is further increased, and the yield of the leguminous plants is increased. Therefore, the invention result can be used for promoting the root nodule formation of the leguminous plants by increasing the fatty acid content of the leguminous plants through genetic engineering or non-genetic engineering.)

1. Use of a fatty acid to promote root nodule formation or increase the number of root nodules in leguminous plants.

2. The use of claim 1, wherein the fatty acids comprise total fatty acids, C14:0, C16:0, C18:2, and/or C18: 3.

3. use of increased fatty acid content in legumes to promote root nodule formation or increase root nodule count in legumes.

4. application of increasing the content of fatty acid in leguminous plants in promoting the nitrogen utilization rate of the leguminous plants.

5. application of increasing the fatty acid content of leguminous plants in increasing the yield of leguminous plants.

6. Use according to any one of claims 3 to 5, wherein the fatty acid content of leguminous plants is increased by genetic engineering.

7. the use of claim 6, wherein the transgenic material of leguminous plants is obtained by genetic transformation of the CeDGAT1 or CectGPDH2 gene derived from Chlorella ellipsoidea,

The nucleotide sequence of the CeDGAT1 is shown as SEQ ID NO. 1; or a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

The nucleotide sequence of the CectGPDH2 gene is shown in SEQ ID NO. 2; or a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2 and expresses the same functional protein.

8. Any one of the following applications of the gene capable of increasing the fatty acid content of leguminous plants,

(1) use in promoting root nodule formation or increasing root nodule number in leguminous plants;

(2) application in promoting nitrogen utilization rate of leguminous plants;

(3) Application in increasing the weight of the hundred grains of leguminous plants;

(4) Application in increasing yield of leguminous plants.

9. The use of claim 8, wherein the gene is the CeDGAT1 or CectGPDH2 gene;

the nucleotide sequence of the CeDGAT1 is shown as SEQ ID NO. 1; or a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

The nucleotide sequence of the CectGPDH2 gene is shown in SEQ ID NO. 2; or a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2 and expresses the same functional protein.

10. Use of a biological material comprising a CeDGAT1 or CectGPDH2 gene in any one of the following applications:

(1) Use in promoting root nodule formation or increasing root nodule number in leguminous plants;

(2) Application in promoting nitrogen utilization rate of leguminous plants;

(3) application in increasing the weight of the hundred grains of leguminous plants;

(4) Application in increasing yield of leguminous plants;

the nucleotide sequence of the CeDGAT11 is shown as SEQ ID NO. 1; or a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

The nucleotide sequence of the CectGPDH2 gene is shown in SEQ ID NO. 2; or a nucleotide sequence which is shown in SEQ ID NO.2 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

The biological material is a recombinant expression vector, an expression cassette, a recombinant bacterium or a host cell.

Technical Field

The invention relates to the technical field of leguminous plant germplasm resource improvement, in particular to application of increasing the content of fatty acid in leguminous plants in promoting the formation of root nodules of the leguminous plants.

background

The root nodule is symbiotic tissue formed by root cortex cell proliferation induced by interaction of rhizobium and leguminous plants. Root nodule tissue can reduce atmospheric nitrogen to ammonia and supply it to its host plant, which is an important strategy for enhanced nitrogen utilization by legumes. Since nodules can increase the efficiency of legumes in utilizing nitrogen, research on the mechanism of nodule formation is a hot spot in legumes today. It is currently believed that nodule formation is regulated primarily by autoregulation of nodule number, development and function on each root, which relies on signal regulation pathways of nitrate-induced long-distance systems. The discovery of the NIN-LIKE PROTEIN transcription factor provides important support for the pathway. KLV down-regulates nodule formation in Lotus japonicus (Lotus japonicus). The number and the size of soybean nodules can be obviously improved by over-expressing GmINS 1. However, no relevant report is found about the relationship between the secondary metabolism of plants and the formation of nodules.

Diacylglycerol O-Acyltransferase (DGAT) produces Triacylglycerol (TAG) by introducing an acyl group at the last carbon position on the carbon backbone of Diacylglycerol (DAG), a rate-limiting enzyme for TAG assembly. Their activity directly affects the synthesis and accumulation of oil in seeds. Due to the increasing demand for vegetable oil, the research on the DGAT gene function of different species is carried out successively. DGAT is considered to be an enzyme that plays a major role in TAG synthesis and is ubiquitous in eukaryotes. DGAT is currently divided into four types: DGAT1, DGAT2, WS/DGAT and intracytoplasmic DGAT3(CytoDGAT), where DGAT1 is the key DGAT for TAG synthesis. The DGAT1 homologous genes of different species are sequentially reported. The heterologous or over-expression of DGAT1 or its mutant in plant can increase the oil accumulation in plant seeds by 11-50%.

Glycerol-3-phosphate (G3P) catalyzed by Glycerol-3-phosphate dehydrogenase (GPDH) is an important raw material for synthesizing Triacylglycerol (TAG), and the enzyme participates in mitochondrial G3P shuttle, provides electrons for respiratory chain, is a key enzyme for G3P synthesis, is also one of key enzymes for connecting sugar metabolism and lipid metabolism, and plays an important role in oil synthesis and energy metabolism in plants. There are three types of GPDH cytoplasmic (ctGPDH), chloroplast (cpgppdh) and mitochondrial (mtGPDH) depending on their subcellular localization. The yeast ctGPDH is transferred into rape, the glycerol content of a transgenic line is 4 times of that of a wild type, the total fatty acid content of seeds is improved by 40 percent and reaches 81.4 +/-10 mg/g fresh weight, the fatty acid composition is not obviously changed, the sucrose content in mature seeds is reduced, but the protein content is not obviously changed.

in 2017, the interaction relationship between plants and symbiotic bacteria is rewritten, and fatty acid is an organic carbon source provided by plants to symbiotic bacteria and is required by fungi to synthesize oil, so that the increase of the fatty acid content of plants is beneficial to the colonization of symbiotic bacteria.

Disclosure of Invention

the invention aims to provide application of leguminous plant fatty acid in promoting nodule formation.

another objective of the present invention is to provide a novel use of the CeDGAT1 or CectGPDH2 gene derived from Chlorella ellipsoidea (Chlorella ellipsoidea).

The invention obtains soybean transgenic material by utilizing a genetic transformation method for CeDGAT1 and CectGPDH2 genes derived from Chlorella ellipsoidea, and researches show that the obtained transgenic soybean does not have undesirable agronomic characters on phenotype; meanwhile, transgenic lines of CeDGAT1 and CectGPDH2 are found, and the hundred grain weight and the total fatty acid in seeds are increased; the number of the root nodules is increased by more than 40 percent; the nitrogen utilization rate is obviously improved. The method is used for improving the content of the soybean fatty acid, promoting the root nodule formation of the leguminous plants, further improving the nitrogen utilization rate of the leguminous plants and realizing the yield increase of the leguminous plants.

In particular, the invention provides the use of fatty acids to promote root nodule formation or increase root nodule count in leguminous plants.

The fatty acids include total fatty acids, C14:0, C16:0, C18:2, and/or C18: 3.

The invention provides an application of increasing the content of fatty acid in leguminous plants in promoting the formation of root nodules or increasing the number of root nodules in leguminous plants.

The invention further provides application of increasing the content of fatty acid in leguminous plants in promoting the nitrogen utilization rate of the leguminous plants.

the invention further provides application of increasing the content of fatty acid of leguminous plants in increasing the yield of the leguminous plants.

The skilled person can increase the fatty acid content of the leguminous plants by any method, preferably by genetic engineering.

In the embodiment of the invention, the gene CeDGAT1 or CectGPDH2 derived from Chlorella ellipsoidea is used for obtaining the transgenic material of leguminous plants by a genetic transformation method,

the nucleotide sequence of the CeDGAT1 is shown as SEQ ID NO. 1; or a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

the nucleotide sequence of the CectGPDH2 gene is shown in SEQ ID NO. 2; or a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2 and expresses the same functional protein.

The invention provides any one of the following applications of a gene capable of increasing the fatty acid content of leguminous plants,

(1) Use in promoting root nodule formation or increasing root nodule number in leguminous plants;

(2) Application in promoting nitrogen utilization rate of leguminous plants;

(3) Application in increasing the weight of the hundred grains of leguminous plants;

(4) Application in increasing yield of leguminous plants.

The skilled person can construct a recombinant expression vector containing a gene capable of increasing the fatty acid content of leguminous plants using an existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. When a gene capable of increasing the fatty acid content of leguminous plants is used for constructing a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added in front of a transcription initiation nucleotide, and can be used independently or combined with other plant promoters; in addition, when the above genes are used to construct recombinant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors used may be processed, for example, by adding genes expressing color-changing enzymes or luminescent compounds in plants, antibiotic markers having resistance, or chemical-resistant marker genes, etc. From the safety of the transgenic plants, the transgenic plants can be directly screened by taking the flowering time as a phenotype without adding any selective marker gene.

in the examples of the present invention, the genes CeDGAT1 and CectGPDH2 derived from chlorella ellipsoidea were selected as genes capable of increasing the fatty acid content of leguminous plants.

The nucleotide sequence of the CeDGAT1 is shown as SEQ ID NO. 1; or a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

the nucleotide sequence of the CectGPDH2 gene is shown in SEQ ID NO. 2; or a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2 and expresses the same functional protein.

The invention provides any one of the following applications of a biological material containing CeDGAT1 or CectGPDH2 genes:

(1) Use in promoting root nodule formation or increasing root nodule number in leguminous plants;

(2) Application in promoting nitrogen utilization rate of leguminous plants;

(3) Application in increasing the weight of the hundred grains of leguminous plants;

(4) Application in increasing yield of leguminous plants;

The nucleotide sequence of the CeDGAT1 is shown as SEQ ID NO. 1; or a nucleotide sequence which is shown in SEQ ID NO.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

The nucleotide sequence of the CectGPDH2 gene is shown in SEQ ID NO. 2; or a nucleotide sequence which is shown in SEQ ID NO.2 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides;

The biological material is a recombinant expression vector, an expression cassette, a recombinant bacterium or a host cell.

The invention obtains soybean transgenic material by utilizing a genetic transformation method for CeDGAT1 and CectGPDH2 genes derived from Chlorella ellipsoidea, and researches show that the obtained transgenic soybean does not have undesirable agronomic characters on phenotype; meanwhile, the transgenic lines of CeDGAT1 and CectGPDH2 are found to have the weight per hundred grains increased by 12.24-25.13% (see A in figure 6) and 8.10-17.03% (see A in figure 7), respectively; the total fatty acids in the seeds were increased by 5.43% -20.97% (see B of fig. 6) and 6.52% -15.01% (see B of fig. 7), respectively; the root nodule number is respectively increased by 44.12-60.66% and 40.69-44.85% (see A of figure 8); the nitrogen utilization rate is respectively improved by 1.94-6.48% and 4.48% -5.86% (see B of figure 8). Neither of CeDGAT1 and CectGPDH2 changed the composition of the fatty acids, but both significantly increased the C14:0, C18:2 and C18:3 content (see C of FIG. 6 and C of FIG. 7). The invention shows that the soybean fatty acid content is increased, the nodule formation of leguminous plants can be promoted, the nitrogen utilization rate of the leguminous plants is further increased, and the yield of the leguminous plants is increased. Therefore, the invention result can be used for promoting the root nodule formation of the leguminous plants by increasing the fatty acid content of the leguminous plants through genetic engineering or non-genetic engineering.

Drawings

Figure 1 is an entry vector for CeDGAT 1.

FIG. 2 is an entry vector for CectGPDH 2.

FIG. 3 is a map of the pHZM06 vector.

Figure 4 is an expression vector for CeDGAT 1.

Fig. 5 is an expression vector for CectGPDH 2.

Fig. 6 shows the effect of heterologously expressed Ce DGAT1 on soybean, a for change in the weight of hundred grains, B for change in total fatty acids, and C for fatty acid composition. Significant differences compared to wild type at p <0.05 levels. Significant differences compared to wild type at p <0.01 levels.

Fig. 7 shows the effect of heterologous expression of CectGPDH2 on soybean, a for change in hundred grain weight, B for change in total fatty acids, and C for fatty acid composition. Significant differences compared to wild type at p <0.05 levels. Significant differences compared to wild type at p <0.01 levels.

FIG. 8 is a graph showing the effect of heterologous expression of CeDGAT1 and CectGPDH2 on soybean nodule formation and nitrogen fixation rate. A, influence on the number of nodules; b, the total nitrogen content of the upper part of the soybean. Significant differences compared to wild type at p <0.05 levels. Significant differences compared to wild type at p <0.01 levels.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The biochemical reagents and materials used in the examples are all commercially available unless otherwise specified. In the experiments in the following examples, three replicates were set up and the results averaged.