Gene OsNST2 for transporting nucleotide sugar and application of gene OsNST2

Abstract

The invention discloses a gene OsNST2 for transporting nucleotide sugar and application of the gene OsNST2. A nucleotide sequence of a coding region of the gene is as shown in SEQ ID No. 1, and an amino acid sequence coded by the gene is as shown in SEQ ID No. 2. It is found for the first time that the growth, development and yield of rice are affected by the OsNST2 by regulating and controlling formation of rice cell walls, an OsNST2 gene knock-down strain is created through an RNAi technology, an OsNST2 gene overexpression strain is obtained through an overexpression transgenic technology, it is verified that the OsNST2 gene is a new gene for regulating and controlling formation of the rice cell walls, and a new gene resource is provided for high yield of rice.

Description

Technical Field

[0001] The present invention belongs to the field of rice molecular breeding and transgenic technology, and specifically relates to a gene for transporting nucleotide sugars. OsNST2 and its applications. Background Art

[0002] As one of my country's most important food crops, rice yield is crucial to national food security. Improving rice yield has always been a key topic in rice breeding. Among the many factors influencing rice yield, biomass is a key factor influencing high rice yield.

[0003] Nucleotide sugars are derivatives of nucleoside diphosphates and monosaccharides. They primarily include 14 types: UDP-α-D-acetylgalactosamine, UDP-α-D-acetylglucosamine, UDP-β-L-rhamnose, UDP-α-D-galactose, UDP-α-D-glucose, UDP-α-D-galacturonic acid, UDP-α-D-glucuronic acid, UDP-β-L-arabinose, UDP-α-D-xylose, UDP-α-D-apiose, GDP-β-L-trehalose, GDP-β-L-glucose, GDP-α-D-mannose, and GDP-β-L-galactose. As key intermediates in plant sugar metabolism, they participate in important physiological processes such as cell wall synthesis, protein glycosylation, and glycolipid synthesis.

[0004] Nucleotide sugar transporters (NSTs) are a class of membrane proteins localized to the Golgi apparatus and endoplasmic reticulum. They possess 6-10 transmembrane domains and play a key role in the biosynthesis of plant cell wall polysaccharides and glycoproteins. NSTs are responsible for transporting nucleotide sugars (such as UDP-glucose and GDP-mannose) from the cytoplasm into the Golgi lumen, where they are synthesized into cell wall matrix polysaccharides (such as hemicellulose and pectin) mediated by glycosyltransferases. Hemicellulose and pectin, as important components of the rice cell wall, regulate rice growth, development, and yield by regulating cell wall formation.

[0005] Existing research indicates that there are over 50 members of the NST family in Arabidopsis thaliana, and several NSTs have been functionally characterized. Functional defects can lead to abnormal cell wall structure, which in turn affects the plant's mechanical strength and growth and development.

[0006] Existing research on NSTs has largely focused on model plants (such as Arabidopsis thaliana), while relevant gene resources in rice are scarce, resulting in a lack of effective targets for crop improvement. Some NSTs specifically recognize and transport a single substrate, while others may recognize and transport multiple different nucleotide sugars. However, the underlying mechanisms for these functions remain unclear.

[0007] The cell wall plays an important role in rice growth, development, and yield formation. Currently, there is a lack of systematic research on the functional specificity, expression regulation mechanisms, and contributions of rice NSTs family members to agronomic traits. Summary of the Invention

[0008] In response to the scarcity of NSTs-related gene resources in rice, the present invention provides a gene for transporting nucleotide sugars. OsNST2 and its application, the present invention first discovered OsNST2 By regulating the formation of rice cell walls, it affects the growth, development and yield of rice, providing new genetic resources for high rice yield.

[0009] In order to achieve the above object, the present invention provides a gene for transporting nucleotide sugars. OsNST2 The nucleotide sequence of its coding region is shown in SEQ ID No.1.

[0010] The amino acid sequence encoded by it is shown in SEQ ID No.2.

[0011] The second aspect of the present invention provides the above-mentioned gene for transporting nucleotide sugars OsNST2 Any of the following applications: (1) Regulate rice cell wall; (2) Used for rice variety improvement; (3) Used to prepare transgenic rice.

[0012] The third aspect of the present invention provides a method for promoting the formation of rice cell walls, which comprises: using genetic engineering means to enhance the OsNST2 Gene.

[0013] Specifically, OsNST2 The gene was used as the target, the primer sequence was designed, the DNA fragment containing the coding primer sequence was connected to the pCAMBIA1300 vector, and the rice was transformed to obtain OsNST2 Transgenic rice with enhanced gene function.

[0014] Specifically, the primer sequence includes the OE-F primer sequence shown in SEQ ID No. 7 and the OE-R primer sequence shown in SEQ ID No. 8.

[0015] The fourth aspect of the present invention provides a method for interfering with rice cell wall formation, comprising: RNAi interference in rice OsNST2 Gene.

[0016] Specifically, the forward interference fragment and the reverse interference fragment were cloned. OsNST2 The fragment of RNAi interference expression of the gene isOsNST2 The gene fragment was connected to the pTCK303 vector and transformed into rice to obtain OsNST2 Transgenic rice with disrupted gene function.

[0017] Specifically, the forward interference fragment primer sequence is shown as SEQ ID No. 3 to SEQ ID No. 4, and the reverse interference fragment primer sequence is shown as SEQ ID No. 5 to SEQ ID No. 6.

[0018] A fifth aspect of the present invention provides the use of the transgenic rice obtained by the above method in plant breeding.

[0019] Through the above technical solution, the present invention achieves the following beneficial effects: The present invention first discovered OsNST2 By regulating the formation of rice cell walls, it affects the growth and yield of rice, and creates a new product through RNAi technology. OsNST2 Gene knockdown strains, obtained through overexpression transgenic technology OsNST2 Gene overexpression lines, verification OsNST2 The gene is a new gene that regulates the formation of rice cell walls and provides a new genetic resource for high rice yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 yes OsNST2 Gene expression level detection results; Figure 2 It is a phenotypic characteristic of rice plants; Figure 3 It is the result of the investigation of agronomic traits of rice plants; Figure 4 It is the result of determination of xylose, galactose, hemicellulose and pectin contents in rice stem cell walls; Figure 5 It is the rice per plant yield phenotype; Figure 6 It is the statistical analysis result of rice single plant yield and plot yield. DETAILED DESCRIPTION

[0021] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0022] Example 1 Analysis of Nucleotide Sugar Transporter Genes OsNST2 The gene is distributed on chromosome 3 of the rice genome and the gene number is LOC_Os03g17740 . OsNST2The nucleotide sequence of the gene coding region is 969 bp long and is shown in SEQ ID No. 1. The amino acid sequence it encodes is 322 amino acids long and is shown in SEQ ID No. 2. The protein is approximately 34.6 kDa in size.

[0023] SEQ ID No. 1: ATGACGGCCAAGGGCGGCGCCGCGCCGTCGCCGGGCGGCGCGGGCGGCCTCCCGAACGGCCGCTTCTTCACCGTGGGGCTCGTCACCGCCTGGTACTCCTCCAACATCGGCGTCCTCCTCCTCAACAAGTACCTCCTCAGCAACTACGGCTTCAAGTACCCAATCTTCCTCACCATGTGCCACATGTCCGCCTGCGCCCTCCTCTCCTACGCCGCCATCGCCTGGCTCCGCGTCGTCCCCATGCAGCTCGTCCGCTCCCGCGTCCAGCTCGCCAAGATCGCCGCCCTCAGCCTCGTCTTCTGCGGCTCCGTCGTCTCCGGCAACGTCTCGCTCCGCTACCTCCCCGTCTCCTTCAACCAGGCCGTCGGCGCCACCACCCCCTTCTTCACCGCCGTCTTCGCCTACATCATGACCGTCAAGAGGGAGTCCTGGGTCACCTACCTCACCCTCGTCCCCGTCGTCACCGGCGTCATGATCGCCAGCGGAGGAGAGCCTAGCTTTCATCTGTTTGGATTCATCATGTGCATTGGGGCCACTGCTGCAAGGGCATTGAAGACGGTGTTGCAAGGAATCCTCCTTTCATCCGAGGGGGAGAAGCTTAATTCTATGAATTTGCTTTTATACATGGCTCCAATTGCTGTTATTCTCTTGCTCCCTGCTACCATCTTCATGGAGGACAACGTTGTTGGCATTACAATAGAACTGGCCAAAAAGGACACCACGATTGTTTGGTTGCTGCTGTTCAATTCCTGTTTGGCATATTTTGTCAATTTGACCAATTTCTTGGTGACTAAGCATACTAGTGCATTGACTCTACAGGTCCTAGGTAACGCGAAAGGTGCTGTGGCAGTTGTTGTCTCAATTCTAATATTCAGGAATCCCGTATCTGTAACCGGAATGCTCGGTTACACTCTTACAGTTATAGGCGTAATTCTTTACAGTGAATCCAAAAAACGCAACAAGCCCTGA。

[0024] SEQ ID No. 2: MTAKGGAAPSPGGAGGLPNGRFFTVGLVTAWYSSNIGVLLLNKYLLSNYGFKYPIFLTMCHMSACALLSYAAIAWLRVVPMQLVRSRVQLAKIAALSLVFCGSVVSGNVSLRYLPVSFNQAVGATTPFFTAVFAYIMTVKRESWVTYLTLVPVVTGVMIAS GGEPSFHLFGFIMCIGATAARALKTVLQGILLSSEGEKLNSMNLLLYMAPIAVILLLPATIFMEDNVVGITIELAKKDTTIVWLLLFNSCLAYFVNLTNFLVTKHTSALTLQVLGNAKGAVAVVVSILIFRNPVSVTGGMLGYTLTVIGVILYSESKKRNKP.

[0025] Example 2 OsNST2 RNAi interference vector and OsNST2 Overexpression vector construction and rice transformation 1. Construction of RNAi Interference Vector The forward interference fragment was cloned using primer sequences such as F1: CGAGCTCtggggccactgctgcaagggca (SEQ ID No. 3), R1: GGACTAGTaccaagaaattggtcaaatt (SEQ ID No. 4) and the reverse interference fragment was cloned using primer sequences such as F2: CGCGGATCCtggggccactgctgcaagggca (SEQ ID No. 5), R2: CGGGGTACCaccaagaaattggtcaaatt (SEQ ID No. 6). OsNST2 The fragment of RNAi interference expression of the gene was digested with Sac1 and Spe1 as well as BamH1 and Kpn1, and the linearized RNAi vector pTCK303 was obtained by gel recovery and purification. OsNST2 The gene fragment was connected to the RNAi vector pTCK303. The above reaction products were transformed into competent E. coli and positive colonies were screened by kanamycin resistance. Then single colonies were selected for shaking and Sanger sequencing verification. OsNST2 The construct was correctly inserted into the pTCK303 vector.

[0026] 2. OsNST2 Overexpression vector construction according to OsNST2The coding region sequence was used to design the overexpression vector primers. The OE-F primer sequence was SEQ ID No.7, such as: ttcgagctcagatctggtaccATGACGGCCAAGGGCGGC, and the OE-R primer sequence was SEQ ID No.8, such as: acgggggactctagtggatccTCAGGGCTTGTTGCGTTTTT. PCR amplification OsNST2 The coding region sequence was recovered by gel fusion OsNST2 The pCAMBIA1300 vector was digested with Kpn1 and BamH1, and the linearized pCAMBIA1300 vector was obtained by gel recovery and purification. OsNST2 The gene fragment was homologously recombined into the overexpression vector pCAMBIA1300. The above reaction products were transformed into competent E. coli and positive colonies were screened by kanamycin resistance. Then single colonies were selected for shaking and Sanger sequencing verification, and finally confirmed. OsNST2 The gene was correctly constructed into the pCAMBIA1300 vector.

[0027] 3. RNAi OsNST2 Gene expression and OsNST2 Overexpression vector rice transformation The constructed pTCK303 and pCAMBIA1300 vectors were transformed into rice callus tissue via Agrobacterium-mediated transfection, using the japonica rice variety Nipponbare as the recipient. Positive transgenic plants were selected using hygromycin, resulting in 20 T0-generation transgenic plants. Rice transformation was performed by Wuhan Boyuan Biotechnology Co., Ltd.

[0028] 4. OsNST2 Identification and phenotypic analysis of gene RNAi interference and overexpression plants 4.1 OsNST2 Gene expression detection: RNA was extracted from leaves of wild-type and transgenic plants and reverse transcribed into cDNA. The sequence of fluorescent quantitative PCR primer qRT-PCR-F is SEQ ID No.9, such as: CTACATCATGACCGTCAAGA, and the sequence of qRT-PCR-R is SEQ ID No.10, such as: ACAGATGAAAGCTAGGCTCT. The results of qRT-PCR experiments showed that compared with the wild-type, the knockdown plants OsNST2 The expression level was significantly reduced, and the overexpression plants OsNST2 The expression level was significantly increased ( Figure 1 ).

[0029] 4.2 OsNST2 Gene RNAi interference and overexpression plant phenotype determination: Wild-type Nipponbare, RNAi interference, and overexpression rice plants were grown in the summer field in Yangzhou, with normal water and fertilizer management. The rice plant phenotypes were photographed ( Figure 2 ) and observed the agronomic traits and found that compared with the wild type, OsNST2 There were no significant changes in the number of ears, number of grains per ear, grain length, grain width and seed setting rate of the RNAi plants, but the plant height, ear length and 1000-grain weight were significantly reduced. OsNST2 There were no significant changes in plant height, grain length, grain width, and seed setting rate of the gene overexpressing plants; however, the ear length, number of ears, number of grains per ear, and 1000-grain weight increased significantly ( Figure 3 ).

[0030] The stems of wild-type, RNAi interference and overexpression plants were further ground into powder, and the content of monosaccharide components and main polysaccharide components of cell walls of plant stems were measured. It was found that: among the monosaccharide components of cell walls, the content of xylose and galactose in the stems of overexpression plants was significantly increased compared with the wild type, while the opposite was true for knockdown plants, but the change in galactose was more significant; among the main polysaccharide components of cell walls, the content of hemicellulose and pectin in the stems of overexpression plants was significantly increased compared with the wild type, while the content of hemicellulose and pectin in knockdown plants was significantly decreased ( Figure 4 ). The results show that OsNST2 The gene may be a xylose and galactose transporter.

[0031] 4.3 OsNST2 Determination of single plant yield and plot yield of gene RNAi interference and overexpression plants: After the seeds matured and were harvested, the harvested grains of each plant were photographed ( Figure 五 ) and the yield of single plant and plot were measured and it was found that compared with the wild-type Nipponbare, the yield of single plant and plot of overexpressed plants increased significantly ( Figure 六 ), increased by 24% and 16% respectively; the yield of single plant and plot of RNAi interference plants was significantly reduced, with a decrease of 15% and 39% respectively.

[0032] The above studies show that OsNST2 This is a new gene that affects cell wall formation by regulating the transport of nucleotide sugars, thereby affecting rice growth, development, and yield formation. Overexpression of this gene can significantly increase rice yield and plays an important role in high rice yield.

[0033] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0034] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0035] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A gene that transports nucleotide sugars OsNST2 , characterized in that, The nucleotide sequence of its coding region is shown in SEQ ID No.

1.

2. The gene for transporting nucleotide sugars according to claim 1 OsNST2 , characterized in that, The amino acid sequence encoded by it is shown in SEQ ID No.

2.

3. The gene for transporting nucleotide sugars according to claim 1 or 2 OsNST2 Any of the following applications: (1) Regulate the formation of rice cell walls; (2) Used for rice variety improvement; (3) Used to prepare transgenic rice.

4. A method for promoting rice cell wall formation, characterized in that: include: Using genetic engineering to enhance the OsNST2 Gene.

5. The method according to claim 4, characterized in that by OsNST2 The gene was used as the target, the primer sequence was designed, the DNA fragment containing the coding primer sequence was connected to the pCAMBIA1300 vector, and the rice was transformed to obtain OsNST2 Transgenic rice with enhanced gene function.

6. The method according to claim 5, wherein The primer sequences include the OE-F primer sequence shown in SEQ ID No. 7 and the OE-R primer sequence shown in SEQ ID No.

8.

7. A method for interfering with rice cell wall formation, characterized in that: include: RNAi interference in rice OsNST2 Gene.

8. The method according to claim 7, characterized in that Cloning with forward interference fragments and reverse interference fragments OsNST2 The fragment of RNAi interference expression of the gene is OsNST2 The gene fragment was connected to the pTCK303 vector and transformed into rice to obtain OsNST2 Transgenic rice with disrupted gene function.

9. The method according to claim 8, wherein The forward interference fragment primer sequence is shown as SEQ ID No.3-SEQ ID No.4, and the reverse interference fragment primer sequence is shown as SEQ ID No.5-SEQ ID No.

6.

10. Use of the transgenic rice obtained by the method according to any one of claims 4 to 9 in plant breeding.

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