Application of the BnaA01.SnRK1.1 gene in Brassica napus

Abstract

This invention discloses the application of the BnaA01.SnRK1.1 gene in Brassica napus. The BnaA01.SnRK1.1 gene is expressed in various tissues, but its expression level is highest in roots, leaves, flowers, and siliques. Its encoded protein is located in the cell membrane and nucleus. Studies have found that overexpression of the BnaA01.SnRK1.1 protein kinase-encoding gene in plants results in shorter and wider siliques, an increase in the number of siliques per plant, and an increase in yield per plant. Therefore, the BnaA01.SnRK1.1 protein kinase can be used to regulate plant architecture and yield. Overexpression of the BnaA01.SnRK1.1 protein in plants can lead to high-yielding rapeseed, which is of great significance for improving crop traits and increasing yield.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the BnaA01.SnRK1.1 gene in Brassica napus. Background Technology

[0002] With global environmental changes, rapeseed production has decreased in most major producing countries, leading to a tightening and persistent shortage of rapeseed globally. Therefore, increasing rapeseed yield has become a primary goal for researchers both domestically and internationally. The harvest index is a crucial indicator in crop science research and production, requiring not only strong photosynthetic assimilation and ample storage space in plants, but also a smooth transport system. In many plant species, sucrose is a vital source of carbon metabolism and a key signaling molecule regulating plant growth and fruit development. SnRK1, an intermediate in the sugar signaling cascade, can influence seed germination and plant growth by regulating sugar metabolism. Sugars are also key components reflecting plant energy status. SnRK1, as a central energy and metabolic sensor in plants, can regulate energy balance to cope with nutrient signals and environmental stresses. Previous studies have found that plants have evolved to modify ancient, highly conserved eukaryotic energy sensors to better adapt to their unique lifestyles and more effectively cope with changing environmental conditions. The α-catalytic subunit of SnRK1 exhibits independent regulatory activity in this process, influencing plant growth, development, and metabolic stress responses. Despite numerous studies on SnRK1, the role of the α subunit as an independently active protein kinase in the growth and development of rapeseed remains unclear. Summary of the Invention

[0003] In view of this, one objective of the present invention is to provide an application of the BnaA01.SnRK1.1 gene of rapeseed in the preparation of rapeseed plants with increased yield; a second objective of the present invention is to provide an application of the BnaA01.SnRK1.1 gene of rapeseed in the preparation of rapeseed plants with increased number of siliques; a third objective of the present invention is to provide an application of the BnaA01.SnRK1.1 gene of rapeseed in the preparation of rapeseed plants with increased number of seeds per silique; a fourth objective of the present invention is to provide an application of the BnaA01.SnRK1.1 gene of rapeseed in the preparation of rapeseed plants with increased number of seeds per silique; a fourth objective of the present invention is to provide an application of the BnaA01.SnRK1.1 gene of rapeseed in the preparation of rapeseed plants with increased length or width of siliques; and a fifth objective of the present invention is to provide a method for increasing rapeseed yield.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] 1. Application of the Brassica napus BnaA01.SnRK1.1 gene in the preparation of single-plant varieties with increased yield, wherein the nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.3.

[0006] Preferably, the plant in this invention is Brassica napus.

[0007] 2. Application of the Brassica napus BnaA01.SnRK1.1 gene in the preparation of plants with increased number of siliques, wherein the nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.3.

[0008] 3. Application of the Brassica napus BnaA01.SnRK1.1 gene in the preparation of rapeseed with increased number of seeds per pod, wherein the nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.3.

[0009] 4. Application of the Brassica napus BnaA01.SnRK1.1 gene in the preparation of siliques that control the length or width of the fruit, wherein the nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.3.

[0010] The preferred embodiment of the present invention is the application of the BnaA01.SnRK1.1 gene of Brassica napus in shortening siliques through overexpression in rapeseed.

[0011] The preferred embodiment of the present invention is the application of the BnaA01.SnRK1.1 gene of Brassica napus in the overexpression of Brassica napus to widen the siliques.

[0012] 5. A method for increasing rapeseed yield, wherein the rapeseed yield is increased by overexpressing the Brassica napus BnaA01.SnRK1.1 gene in rapeseed, and the nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO. 3.

[0013] Preferably, the rapeseed in this invention is Brassica napus.

[0014] The beneficial effects of this invention are as follows: This invention discloses the application of the BnaA01.SnRK1.1 gene in Brassica napus. Studies have found that the SnRK1.1 protein kinase can regulate plant yield. GUS histochemical staining revealed that BnaA01.SnRK1.1 is expressed in various tissues, with higher expression levels in leaves, roots, anthers, calyxes, and siliques, and lower expression levels in siliques and seeds. A transient transformation method in tobacco was used to investigate the localization of the BnaA01.SnRK1.1 protein in the cell membrane and nucleus. Agronomical trait study of transgenic rapeseed showed that, compared with the control plants, the yield per plant of overexpressing transgenic rapeseed was (8.80±1.38 g, 9.4±0.78 g). The average weight gain was 39.61% higher than that of the wild type (6.52±0.65g); the average number of siliques per plant (258±19, 287±26) was 30.88% higher than that of the wild type (208±1); the average number of seeds per silique (28.83±0.24, 29.17±0.24) was 6.88% higher than that of the wild type (27.17±0.24); the average length of the silique (6.0±0.04, 5.96±0.008cm) was 44.55% lower than that of the wild type (10.79±0.05cm); and the average width of the silique (6.8±0.007, 6.67±0.039mm) was 47.18% higher than that of the wild type (4.58±0.017mm). The results of these agronomic trait surveys indicate that overexpression of Brassica napus BnaA01.SnRK1.1 can significantly alter the yield per plant, the number of siliques per plant, the length of the siliques, and the number of grains per silique, which is of great significance for increasing rapeseed yield. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0016] Figure 1 The expression of the BnaA01.SnRK1.1 promoter in Arabidopsis thaliana is shown (ae represents the expression in the whole plant, roots, and leaves during the vegetative growth period of Arabidopsis thaliana, and fj represents the expression in the stem, flower, seed, and silique pericarp after bolting. The scale bar is 2 mm).

[0017] Figure 2 Subcellular localization of BnaA01.SnRK1.1 (GFP represents green excitation light state, Bright represents bright field, mCherry represents chloroplast autofluorescence, Merge represents superposition state, scale bar length is 10 μm);

[0018] Figure 3 Phenotypic observation during the vegetative growth stages of transgenic plants (A: 90 days of field growth and budding stage of rapeseed; B: Detection of transgenic line expression level; C: Detection of net photosynthetic rate of plants, with a scale bar length of 5 cm);

[0019] Figure 4 Phenotypic observation during the reproductive growth period of transgenic plants (A: morphological observation of rapeseed main inflorescence buds; B: morphological observation of seeds after 15 days of growth; C: siliques after 40 days of growth; D: statistical data of siliques after 40 days of growth, from left to right: silique length, silique width, and number of seeds per silique, with a scale bar length of 2 mm).

[0020] Figure 5 For the investigation of agronomic traits of transgenic plants at maturity (A: morphological observation of mature plants; B: investigation of agronomic traits of mature plants, b1-b3 are plant height, first cotyledon circumference, and first branch height, respectively; b2-b4 are the number of siliques per plant, aboveground dry weight, and grain yield per plant, respectively; the scale bar is 5cm). Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0022] The materials used in this invention are as follows: wild-type Brassica napus ZS11, transgenic Brassica napus overexpressing BnaA01.SnRK1.1, Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 strains, and vectors pEarleyGate101 and pCAMBIA1305.1.

[0023] Example 1: Cloning and expression of the BnaA01.SnRK1.1 gene

[0024] Total RNA was extracted from rapeseed using the EZ-10 DNA away RNA Mini-Preps Kit according to the instructions. The quality and concentration of RNA samples were detected by 1% agarose gel electrophoresis. cDNA was synthesized using the reverse transcription kit from US EVERBRIGHT INC.

[0025] Using the rapeseed genome website Brassica napus database ( http: / / www.genoscope.cns.fr / brassicanapus / Obtain the sequences of SnRK1.1 family members, and design specific amplification primers based on the ORF sequence of the BnaA01.SnRK1.1 gene. The specific primers are as follows:

[0026] Upstream primer: 5'-atggatggatcaggaggcggtag-3' (SEQ ID NO.1);

[0027] Downstream primer: 5'-tcagaggactcggagctgagcaag-3' (SEQ ID NO.2);

[0028] Using the extracted cDNA as a template, PCR amplification was performed. The amplification product was sequenced to obtain a 1536 bp cDNA sequence, as shown in SEQ ID NO.3.

[0029] Using the pENTR / D-TOPO Cloning Kit (K240020), the amplified BnaA01.SnRK1.1 gene cDNA was recombined into the pENTR / D-TOPO entry vector via attL1 and attL2 sites, and then transformed into competent E. coli DH5α cells. After identifying positive clones using primers SEQ ID NO.1 and SEQ ID NO.2, plasmids were extracted using the EasyPure Plasmid MiniPrep Kit and subjected to an LR reaction with the pEarleyGate101 expression vector, allowing the target gene to recombine via attR1 and attR2 sites, forming the pEarleyGate101-BnaA01.SnRK1.1 vector for overexpression and subcellular localization.

[0030] Based on the promoter sequence of the BnaA01.SnRK1.1 gene, specific primers were designed:

[0031] Upstream primer: 5'-atatccatgtaaattatatgaaacag-3' (SEQ ID NO.4);

[0032] Downstream primer: 5'-tttctacacaaagagctaataatatt-3' (SEQ ID NO.5);

[0033] The promoter sequence 1993 bp upstream of the gene was amplified using SEQ ID NO.4 and SEQ ID NO.5, as shown in SEQ ID NO.6. After amplification using recombinant primers, it was fused into the pCAMBIA1305.1 vector between the BamH1 and Pst1 restriction sites using recombination. The recombinant primer sequences are as follows:

[0034] Upstream primer: 5'-gagctcggtacccggggatccggatcctactttaatatccatgtaaattatat-3' (SEQ ID NO.7);

[0035] Downstream primer: 5'-gccaagcttgcatgcctgcagctgcagtttctacacaaagagctaataa-3' (SEQ ID NO.8);

[0036] A recombinant plasmid pCAMBIA1305.1-BnaA01.SnRK1.1, containing a promoter fused with the GUS reporter gene, was obtained. This plasmid was transformed into Agrobacterium GV3101 and then infected with wild-type Arabidopsis thaliana to obtain T2 generation transgenic plants. Histochemical staining was used to stain these plants, and the results are as follows: Figure 1 As shown in the figure. The results showed that BnaA01.SnRK1.1 has constitutive expression characteristics, and it is expressed in various parts of the plant at different stages, but the expression levels vary. During the vegetative growth stage, high expression is observed in leaves and roots; during the reproductive growth stage, higher expression levels are observed in leaves, epidermal hairs, flowers, and siliques.

[0037] To determine the intracellular expression location of BnaA01.SnRK1.1 protein, the expression of BnaA01.SnRK1.1 was determined by the localization of the fluorescent protein, utilizing the characteristic of the fused yellow protein marker gene YFP on the pEarleyGate101-BnaA01.SnRK1.1 vector. The results are as follows: Figure 2 As shown in the figure. The results indicate that the BnaA01.SnRK1.1 protein is simultaneously localized in the cell nucleus and cell membrane.

[0038] Example 2: Effects of BnaA01.SnRK1.1 on rapeseed growth

[0039] To investigate the biological function of BnaA01.SnRK1.1 in rapeseed, pEarleyGate101-BnaA01.SnRK1.1 was transformed into the rapeseed variety ZS11 (wild-type, WT) using Agrobacterium-mediated hypocotyl infection, resulting in transgenic lines with significantly increased expression levels. The growth of rapeseed overexpressing BnaA01.SnRK1.1 and wild-type rapeseed was observed after 90 days of growth under the same conditions. The results are as follows: Figure 3 As shown. There was no significant difference in size between the overexpressing plants, but the leaves were rounder and more compact. When they grew to the budding stage, the BnaA01SnRK1.1 overexpressing plants were more upright, with rounder leaf lobes, and the central and basal leaves were similar in shape and thicker.

[0040] The phenotypes of plants in the field during their reproductive growth period were observed, and the results were as follows: Figure 4As shown in the figure. The results showed that, during the full bloom period, compared with the wild type, the inflorescences of plants overexpressing BnaA01.SnRK1.1 were more compact and the flower bud density was increased. Observation of silique growth after flowering revealed that, compared with the wild type, the length of siliques in plants overexpressing BnaA01.SnRK1.1 began to differ from day 10, and this difference persisted until maturity, with the siliques becoming wider. When the siliques of Brassica napus reached 15 days of growth, the growth rate was the highest and seeds began to develop. Observation of seed distribution and morphology in the siliques at this time showed that BnaA01.SnRK1.1 overexpression resulted in a denser seed distribution than WT, but the seeds were smaller. Statistical analysis of silique length, width, and number of seeds per silique after approximately 40 days of growth revealed that the siliques of the overexpressing rapeseed were significantly shorter than those of the wild type, but the width was significantly increased, and the number of seeds per silique was increased.

[0041] Example 3: Investigation of agronomic traits at maturity of rapeseed overexpressing BnaA01.SnRK1.1

[0042] The characteristics of mature plants were investigated and statistically analyzed, and the results are as follows: Figure 5 As shown in the figure. The results showed that the first cotyledon circumference of plants overexpressing BnaA01.SnRK1.1 was slightly larger than that of the wild type, indicating that BnaA01.SnRK1.1 overexpression leads to thicker rapeseed stems. After BnaA01.SnRK1.1 overexpression, plant height and the height of the first effective branch increased, the inflorescences became more compact, the number of flower buds increased, and the number of siliques per plant increased at the final maturity stage. Statistical analysis of the whole rapeseed plant traits revealed that BnaA01.SnRK1.1 overexpression significantly increased the aboveground dry weight and grain yield of rapeseed compared to the wild type.

[0043] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of the BnaA01.SnRK1.1 gene in rapeseed (Brassica napus) to increase the yield of a single rapeseed plant, characterized by: The Brassica napus BnaA01.SnRK1.1 gene is overexpressed in rapeseed to increase the yield of rapeseed plants. The nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that: The rapeseed in question is Brassica napus.

3. The application of the BnaA01.SnRK1.1 gene in increasing the number of siliques per plant in rapeseed, characterized by: The Brassica napus BnaA01.SnRK1.1 gene is overexpressed in rapeseed to increase the number of siliques per plant. The nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.

3.

4. The application of the BnaA01.SnRK1.1 gene in increasing the number of seeds per pod in rapeseed, characterized by: The Brassica napus BnaA01.SnRK1.1 gene is overexpressed in rapeseed to increase the number of seeds per pod. The nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.

3.

5. Application of the BnaA01.SnRK1.1 gene in controlling the length and width of rapeseed siliques, characterized by: Overexpression of the Brassica napus BnaA01.SnRK1.1 gene in rapeseed results in shorter siliques and wider siliques. The nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.

3.

6. A method for increasing rapeseed yield, characterized in that: Rapeseed with increased yield was obtained by overexpressing the Brassica napus BnaA01.SnRK1.1 gene in rapeseed. The nucleotide sequence of the Brassica napus BnaA01.SnRK1.1 gene is shown in SEQ ID NO.

3.

7. The method according to claim 6, characterized in that: The rapeseed in question is Brassica napus.

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