Cloning and application of a soybean growth-promoting gene GmGRP1

Abstract

The invention discloses a gene capable of promoting soybean lateral root growth, nodulation and nitrogen fixation and soybean aerial part growth GmGRP1 Cloning and application of the gene GmGRP1 It is cloned from an atypical MYB-CC transcription factor that is highly expressed in soybean lateral root tips and nodules. GmGRP1 (Plant Growth Protein) significantly increased the density of soybean secondary lateral roots, overexpression GmGRP1 It also promoted the number of nodules in soybean, and finally overexpressed GmGRP1 It promotes the growth of the aboveground part of soybeans, increases plant height and number of nodes, increases the number of pods per plant and grain weight per plant, and ultimately increases the yield of soybeans per plant.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a gene that promotes soybean growth GmGRP1 cloning and application. Background Art

[0002] The rapid growth and development of plants requires the optimization and integration of photosynthetic carbon fixation (photosynthesis) in the aboveground part and the absorption of mineral nutrients from the soil by the underground roots. For soybeans, a leguminous plant, it can also provide a considerable amount of nitrogen source for its growth and development through symbiotic nitrogen fixation with rhizobia. Research and discovery of methods that can synergistically regulate the growth of the underground roots of soybeans, increase the absorption of mineral nutrients, and simultaneously regulate the growth of the aboveground part of soybeans, improve the photosynthesis capacity, and promote the growth of soybeans have important theoretical and potential application value. The present invention provides a gene that can significantly promote the density of soybean secondary lateral roots, promote soybean nodulation and nitrogen fixation, and ultimately improve the absorption of nitrogen and phosphorus in soybeans. GmGRP1 It was also found that overexpression GmGRP1 The gene significantly promotes the growth of the aboveground part of soybeans, including increasing plant height and number of nodes, and ultimately increasing the number of pods per plant, the number of grains per plant and the weight of grains per plant, thereby increasing soybean yield. Summary of the invention

[0003] The purpose of the present invention is to provide a method for cloning and applying a gene plant that promotes soybean growth.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] GmGRP1 Application of the gene and / or its encoded protein in promoting soybean growth, the GmGRP1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0006] Furthermore, the soybean growth promotion is embodied in at least one of the following aspects 1)-7):

[0007] 1) Increase the density of secondary lateral roots;

[0008] 2) Increase the number of nodules;

[0009] 3) Increase nodule weight;

[0010] 4) Increase plant height;

[0011] 5) Increase the number of sections;

[0012] 6) Increase nitrogen absorption;

[0013] 7) Increase phosphorus absorption.

[0014] GmGRP1 Application of the gene and / or its encoded protein in increasing soybean yield, the GmGRP1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0015] Furthermore, the improvement of soybean yield is embodied in at least one of the following aspects 1)-3):

[0016] 1) Increase the number of pods per plant;

[0017] 2) Increase the number of seeds per plant;

[0018] 3) Increase seed weight.

[0019] The significant advantages of the present invention are:

[0020] The present invention provides a gene for promoting soybean growth GmGRP1 By overexpression GmGRP1 The gene can simultaneously promote the growth and development of the soybean root system and the aboveground part, which is ultimately beneficial to increasing the yield of the entire soybean plant. GmGRP1 The gene can significantly promote the development of soybean lateral roots, increase lateral root density, increase the number of soybean nodules, and improve the ability of soybean to obtain nitrogen and phosphorus; secondly, overexpression GmGRP1 The gene can significantly promote the growth and development of the aboveground part of soybeans, which is manifested in increasing soybean plant height, increasing the number of soybean nodes, increasing the number of pods and grains per soybean plant and the grain weight per soybean plant, ultimately increasing soybean yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : GmGRP1 Gene cloning and sequence analysis.

[0022] Figure 2 : Overexpression GmGRP1 Effects on the aboveground part and secondary lateral root density of soybean.

[0023] Figure 3 : Overexpression GmGRP1 Effects on soybean growth and nodulation.

[0024] Figure 4 : Overexpression GmGRP1 Effects on nitrogen and phosphorus nutrient absorption of soybean.

[0025] Figure 5 : Overexpression GmGRP1 Effects on soybean yield traits. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments.

[0027] In the following examples, operations not described in detail are all routine biological experimental operations, which can be performed with reference to molecular biology experimental manuals and existing published journals and literature.

[0028] Example 1

[0029] (1) Gene cloning and functional studies

[0030] The soybean root system (nodules) of the soybean variety William 82 was harvested, ground with liquid nitrogen, 500 µL of powder was taken, clean EP was added, 1 mL of Trizol reagent was added, and the mixture was shaken, then placed on ice for 10 min, 200 µL of chloroform was added, and the mixture was shaken for 2 min, then placed on ice for 10 min, centrifuged at 12000 rpm for 15 min, the supernatant was taken, 500 µL of ice isopropanol was added, the mixture was inverted 5-10 times, and centrifuged at 12000 rpm for 15 min. The supernatant was discarded, and the precipitate was washed 2-3 times with 75% ethanol, then blown dry, and the precipitate was dissolved with sterile water to determine the RNA concentration. The root (nodule) samples were reverse transcribed using the reverse transcription kit (TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix), and the steps were performed according to the instructions. The reverse transcribed cDNA was used GmGRP1 Specific primers ( GmGRP1-PCR-F :5'-ATGGAGGGGTGGTGGAAGAG-3'; GmGRP1-PCR-R :5'-TTATACCCTATATGATGAAACC-3') for PCR amplification. The total volume of the PCR reaction system was 50 µL: cDNA 4 µL, forward and reverse primers 2 µL each, Tap enzyme 2 µL, dNTP 5 µL, 10 x PCR Buffer 5 µL, water 35 µL. The PCR reaction program was: 95℃, 5 min, 95℃, 30 s, 60℃ 30 s, 68℃, 60 s, 35 cycles, 68℃, 5 min. The amplified product was detected by agarose gel electrophoresis, and the product size was 813 pb, which was consistent with the expected size ( Figure 1 A). The PCR product was purified and recovered (EasyPure Quick Gel Extraction Kit), connected to the T-blunt vector (purchased from Quanshijin), and sequenced. GmGRP1 The gene sequence results are shown in Figure 1 B. According to GmGRP1 The nucleotide sequence of the gene was used to deduce the encoded GmGRP1 protein sequence, and the protein domain was predicted. The results showed that ( Figure 1C) GmGRP1 protein contains a MYB-CC domain and encodes a transcription factor protein.

[0031] For research GmGRP1 The function of genes, using transgenic technology, GmGRP1 The gene was placed behind the plant strong promoter 35S to construct an overexpression vector (with pFGC5941 vector as the backbone). The overexpression vector was transformed into Agrobacterium ( EHA105 )-mediated method, through callus transformation, GmGRP1 The gene was overexpressed in soybean plants. The results of transgenic identification showed that compared with wild-type plants (wild-type Williams 82 soybean), the overexpressed transgenic plants GmGRP1 The transcription level of the gene increased 40-52 times in leaves ( Figure 2 A), the plant height and number of nodes of transgenic soybean seedlings were significantly higher than those of wild-type soybeans ( Figure 2 B), indicating overexpression GmGRP1 The gene has the potential to promote soybean growth. GmGRP1 The effect of genes on soybean root growth was studied by planting wild-type soybeans and two independent strains of transgenic soybeans in hydroponic nutrient solution. The characteristics of soybean roots were statistically analyzed 15 days after seedling emergence. GmGRP1 Significantly increased the density of soybean secondary lateral roots (about 12%) ( Figure 2 C, Figure 2 D).

[0032] Secondly, wild-type soybeans and transgenic soybeans were planted in soil for one month, and the growth of wild-type soybeans and overexpressed transgenic soybeans was compared. GmGRP1 Gene significantly boosts soybean growth Figure 3 A). Counting the number of root nodules of wild-type soybean and two transgenic lines showed that overexpression GmGRP1 The number of nodules (increased by 67%-70%) and weight of nodules (increased by 33%-40%) of transgenic plants expressing the gene were significantly higher than those of wild-type soybeans, indicating that overexpression of GmGRP1 Promote soybean nodulation ( Figure 3 B, Figure 3 C, Figure 3 D). Further analysis of the nitrogen, phosphorus and potassium nutrient content of wild-type soybeans and overexpressed transgenic soybeans showed that compared with wild-type soybeans, GmGRP1 The nitrogen (increased by 17%-32%) and phosphorus (increased by 10%-21%) contents of the overexpressed transgenic soybeans were significantly higher than those of the wild-type soybeans, while there was no significant difference in potassium content between the two. Figure 4 ), indicating overexpression GmGRP1It can significantly increase soybean's acquisition of nitrogen and phosphorus.

[0033] Example 2

[0034] To further investigate the overexpression GmGRP1 A field experiment was conducted to study the effects of soybean growth and yield under field conditions. The field experiment design is as follows (experimental location: Experimental base of the Institute of Grain and Oils, Hebei Academy of Agricultural Sciences): soybean plant spacing is 20 cm, row spacing is 40 cm, 3 seeds are sown in each hole (finally 2 plants are left in each hole), each plot has a row length of 6 m, and 3 plots of each genotype of soybean are planted. Water and fertilizer management during soybean growth follows the local soybean planting management model. Soybeans are harvested at maturity. The growth of wild-type and transgenic soybeans in the field is as follows: Figure 5 The plant phenotype is shown in Figure 5 As shown in A. At the full maturity stage, 4 plants were randomly harvested from each plot, and the number of pods, seed grains and grain weight per plant were measured for each soybean line. The results show ( Figure 5 B, Figure 5 C, Figure 5 D): Overexpression GmGRP1 The number of pods, number of seeds per plant, and grain weight per plant of transgenic soybeans were significantly higher than those of wild-type soybeans; the number of pods increased by 19%-20%, the number of seeds per plant increased by 12%-17%, and the grain weight per plant increased by 9%-15%. GmGRP1 The gene can significantly promote soybean growth and increase soybean yield, and has good application potential.

Claims

1. Overexpression GmGRP1 The use of a gene and / or its encoded protein in promoting soybean growth is characterized by: The GmGRP1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmGRP1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; The soybean growth promotion is embodied in at least one of the following aspects 1) to 7): 1) Increase the density of secondary lateral roots; 2) Increase the number of nodules; 3) Increase nodule weight; 4) Increase plant height; 5) Increase the number of sections; 6) Increase nitrogen absorption; 7) Increase phosphorus absorption.

2. Overexpression GmGRP1 The application of the gene and / or its encoded protein in increasing soybean yield is characterized by: The GmGRP1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmGRP1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; The improvement of soybean yield is embodied in at least one of the following aspects 1)-3): 1) Increase the number of pods per plant; 2) Increase the number of seeds per plant; 3) Increase seed weight.

Images