Gene for regulating and controlling thickness of flag leaf and stem of wheat and application of gene

Abstract

The invention discloses a gene for regulating and controlling the thickness of flag leaves and stalks of wheat and application of the gene, and belongs to the technical field of plant genetic engineering. The full-length nucleotide sequence of the RLK4DS-2 gene disclosed by the invention is as shown in SEQ ID NO. 1. According to the invention, the RLK4DS-2 gene is cloned from the 4D chromosome of aegilops tauschii, and the influence of the RLK4DS-2 gene on the thickness of flag leaves and stalks of wheat is verified by utilizing a wheat stable genetic transformation experiment, so that a theoretical basis is provided for establishing a morphological marker by utilizing the fine stalk RLK4DS-2 gene and assisting a wheat crop molecular marker breeding and cross breeding system.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a receptor-like protein kinase gene for regulating the thickness of wheat flag leaves and stems and an application thereof. Background Art

[0002] Receptor-Like Protein Kinase (RLKs) is a common type of protein kinase in plants. It plays an important role in signal recognition and transmission, and is involved in plant growth and development, morphological construction, resistance to pathogens, and response to various biological or abiotic stresses. Its gene family is huge and can be divided into six subfamilies. Most of the RLKs identified in plants belong to the leucine-rich repeat sequence type (LRR-RLKs). LRR-RLKs can act as signal recognition receptors in different plants to participate in signal transduction, and play an important role in plant growth and development, stress response, and signal conversion processes.

[0003] Agronomic traits of wheat include plant height, tillering and stem diameter. The cloned genes Rht1, Rht2, Rht8 and Rht24 are all genes that control wheat plant height, which can reduce wheat plant height and increase wheat's ability to resist lodging (Van DeVelde K, Thomas SG, Heyse F, Kaspar R, Van Der Straeten D, Rohde A. (2021) N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles. Molecular plant 14: 679-687; Chai L, Xin M, Dong C, Chen Z, Zhai H, Zhuang J, Cheng X, Wang N, Geng J, Wang X, Bian R, Yao Y, Guo W, Hu Z, Peng H, Bai G, Sun Q, Su Z, Liu J, Ni Z. (2022) A natural variation in Ribonuclease H-like gene underlies Rht8 to confer "GreenRevolution" trait in wheat.Molecular Plant 15:377-380; Tian X,Xia S. (2022) Rht24b, an ancient variation of TaGA2ox-A9, reduces plant height without yieldpenalty in wheat. New phytologist 233:738-750).The gene TN1, which controls wheat tillering, regulates the molecular mechanism of wheat tillering by affecting abscisic acid (ABA) synthesis and signal transduction pathways, providing important genetic resources and theoretical support for wheat plant type improvement (Dong C, Zhang L, Zhang Q, Yang Y, Li D, Xie Z, Cui G, Chen Y, WuL, Li Z, Liu G, Zhang X, Liu C, Chu J, Zhao G, Xia C, Jia J, Sun J, Kong X, Liu X. (2023) Tiller Number 1 encodes an ankyrin repeat protein that controls tillering inbread wheat. Nature communications 14: 836). Studies have found that TaHST1L mediates the auxin signal transduction pathway by interacting with TaIAA17, regulating the level of endogenous auxin, thereby affecting the tillering pattern of wheat (Zhao L, Zheng Y, Wang Y, Wang S, Wang T, Wang C, Chen Y, Zhang K, Zhang N, Dong Z, Chen F. (2023) A HST1-like gene controls tiller angle through regulating endogenous auxin in common wheat. Plant biotechnology journal 21: 122-135).

[0004] The gene NnXTH23 that controls xyloglucan endotransglycosidase / hydrolase in lotus of the Nymphaeaceae family plays an important role in regulating plant height and stem thickness. After the overexpression vector constructed by heterologous transformation was transformed into rapeseed, the plant height and stem thickness of the overexpressed rapeseed were significantly improved. The SD1 gene of tomato positively regulates the development of tomato stems by regulating the size and number of secondary phloem cells (Ye J, Meng XF, Tao PW, Li CX, Liu GZ, Chen WF, Wang Y, Li HX, Ye ZB, Zhang YY. (2020) Tomato SD1, encoding a kinase-interacting protein, is a major locus controlling stem development. Journal of Experimental Botany 71: 3575-3587). In rice, it was found that the OsIPA1 / OsSPL14 gene affects the development of the stem. When the rice OSIPAI / OSSPL14 gene is overexpressed, the rice stem becomes thicker, while when the expression of the OsIPA1 / OsSPL14 gene is downregulated, the stem of the rice plant becomes thinner (Miura K, Ikeda M, Matsubara A, Song XJ, Ito M, Asano K, Matsuoka M, Kitano H, Ashikari M. (2010) OsSPL14 promotes panicle branching and higher grain productivity in rice. Nature genetics 42: 545-549).

[0005] Slender wheat is a genetically modified wheat variety that has a thinner stem. This trait can be applied to wheat breeding. There are few reports on genes that control wheat stem thickness. Three stem thickness QTLs were detected on chromosomes 3A and 4D in the winter wheat DH population (Berry PM, Berry ST. (2015) Understanding the genetic control of lodging-associated plant characters in winter wheat (Triticum aestivum L.). Euphytica 205: 671-689). QTL mapping for stem diameter traits was performed on the RIL population of the hybrid of Kenong 9204 and Jing 411, and a total of 47 additive QTLs related to stem diameter were detected (Yu HT, Liu YN, Zou ZF, Sun XS, Zhang JS, Song S, Wang LJ, Qin R, Sun H, Cui F, Zhao CH, Shi SB. (2023) QTL detection for internode diameter and its association with yieldrelated traits in wheat. Cereal Research Communications 51: 101-113).

[0006] As one of the most important food crops in my country, increasing wheat yield by regulating wheat plant height, tillering and stem thickness is of great significance to ensuring grain production. However, in the current existing technology, there are no genes and applications that can simultaneously regulate the thickness of wheat flag leaves and stems. The genes related to regulating flag leaves and plant type in wheat are still relatively limited, and the related regulatory mechanisms and action pathways need to be further developed and analyzed. Summary of the invention

[0007] In view of the above prior art, the present invention aims to provide a gene for regulating the thickness of flag leaves and stems of wheat and its application. The present invention identified RLK on chromosome 4D of Aegilops tauschii 4DS-2 Gene, and the RLK was verified by wheat stable genetic transformation experiment 4DS-2 The effect of genes on the thickness of wheat flag leaves and stems provides a basis for the future use of thin-stem RLK 4DS-2 Genes provide a theoretical basis for establishing morphological markers and assisting molecular marker breeding and hybrid breeding systems for wheat crops.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a gene RLK for regulating the thickness of wheat flag leaves and stems by Aegilops truncatus 4DS-2 The RLK 4DS-2 The full-length nucleotide sequence of the gene is shown in SEQ ID NO.1; the RLK 4DS-2 The CDs nucleotide sequence of the gene is shown in SEQ ID NO.2; the RLK 4DS-2 The amino acid sequence of the gene is shown in SEQ ID NO.3.

[0010] Beneficial effects of the present invention:

[0011] The present invention cloned RLK on chromosome 4D of Aegilops tauschii 4DS-2 Gene; and used wheat stable genetic transformation experiments to verify RLK 4DS-2 The effect of the gene on the thickness of wheat flag leaves and stems; positive transgenic wheat was screened, and the transgenic positive wheat plants showed a phenotype of thin leaves and stems. The statistical results showed that compared with the transgenic negative control, the width of the flag leaves of the positive wheat plants was reduced by 126%, the length did not change significantly, and the stem diameter was reduced by 98%. 4DS-2 Genes provide a theoretical basis for establishing morphological markers and assisting molecular marker breeding and hybrid breeding systems for wheat crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown is a characteristic schematic diagram of the vector PUbi::RLK4DS-2 in Example 3;

[0013] Figure 2 Shown is a schematic diagram of the positive screening of 186-RLK4DS-2 in Example 3;

[0014] Figure 3 The phenotype of the positive plants of 186-RLK4DS-2 transgenic wheat in Example 3 is shown. DETAILED DESCRIPTION

[0015] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0016] In the early stage of this study, a stripe rust resistance gene cluster was discovered from a 39 kb segment on Aegilops truncatus 4DS. This segment contains a total of 5 genes, namely, receptor-like protein kinase gene RLK 4DS-1 , RLK 4DS-2 , NLR gene NLR 4DS-1, and two potential genes predicted, temporarily named unknown1 and unknown2. The BAC library was further used to clone the RLK 4DS-2 Gene.

[0017] The present invention is directed to RLK 4DS-2 Gene overexpression vector PC186-RLK 4DS-2 (PUbi::RLK 4DS-2 ), wheat was stably transformed by Agrobacterium-mediated genetic transformation, and transgenic positive wheat plants showed a phenotype of thin leaves and stems. The thin leaves and stems phenotype can be directly observed with the naked eye during the adult stage without the need for molecular marker screening. 4DS-2 Genes establish morphological markers to assist in molecular marker breeding and hybrid breeding systems for wheat crops.

[0018] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0019] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. The present invention introduces the expression vector into plant cells, and the introduction methods are well known to those skilled in the art, including but not limited to: Agrobacterium-mediated genetic transformation. If the specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are usually followed, such as J. Sambrook et al., ed., Science Press, 2002, Molecular Cloning Experiment Guide (3rd Edition); DL Spector et al., ed., Science Press, 2001, Cell Experiment Guide; or the conditions recommended by the manufacturer.

[0020] Example 1: Aegilops tauschii RLK 4DS-2 Gene cloning

[0021] Using the diploid Aegilops tauschii material PI511383, we designed RLK based on the candidate gene sequences. 4DS-2 The full-length cloning primers (Table 1) were used to successfully clone RLK 4DS-2 Full-length gene sequence (SEQ ID NO.1).

[0022] Table 1 RLK 4DS-2 Gene cloning and transgenic screening primer sequences

[0023]

[0024] RLK 4DS-2 Full-length gene sequence (SEQ ID NO.1):

[0025] ATGCTAACGAGCCGGTGCGTATTCATCACAGCGGTGCTGCAACTACTAATCTCCTTCCTGGCGGTCGCCGGGGAACCACAAATCGGGCTGCCCGGCTGCCAGACCCGCTGCGGCGACGTGAGCGTGTCGTACCCGTTCGGCATGGGACCCAAGAAGTGCTACTGGCCGGGTTTGAAGCTCACCTGCGACGACCGCGGAAGCAAACCCCCGCCCAGTATTTCGCCGCAGACTACAACTCCGACAGCTGCTCCAACTTGGGCTGCTGCCAGTCGTCCATCTTGTCCAGCAGCACGTCCTATGATGTCGAGGTCAATCGGCTCGGCGATTGCCTGCGGTACGGCCCTGTGCCCGACCCGTTTCCCGACGATGTTCCGGTGAATGTGCTCATCGCTGAGAAGGGTTGGTTCTACCAGGAGTGGTCACGCAATGTCATAAATGTTACCTCGCGGCCCACCGTGATTTTCGAGTGGCGGGTGCCGCTTGGTGCCCCGGTCCCGGTGGGGCCGTGCCCCGACGACGCAGCCCGGAGCATCTGCAAGAGCACCCATAGCTATTGCACACAAGAGTGGTCAAATAATACAGTCGGGGCCTATTCTTGCCAGTGCAACTCCGGCTACACCGGCAACCCTTACCTCACCGATGGATGCCAAG GTGAGTTCACAACTTCACATGTGCACATGTCCAT GTTTTGTTAACTTAATTACAGTATCTTTTATTTACTTAGTCTTAAACAAGACATGCTAAACTATATTGTGTGCAG ATATCCATGTGTGTGATCAGAAAGAAAAACATGGCTGCTTCGGCCATTGTGAGCGACTGCCCGGATATGTTAGCTGCCGGTGCCCAGAAGGAACCCATGGCAACTACTCCATACCCGGGGGCTGTGTCGCAGACACAAACACA GGTATGACACGCA CCAGCCAGTCGATTACTACTACTAGTACGTGCAAACAATGGAACTGTTATTTTAATGGGCCTGCTTAAGCAATTAGT TATAGTAAACATATGTGCGTGTATTGTTTAACTTTCAG

[0026] Note: The underlined region in the sequence is the intron sequence.

[0027] Example 2: RLK 4DS-2 Gene sequence modification

[0028] The cloned RLK 4DS-2 The full-length gene sequence was compared and analyzed in NCBI, and it was found that the full-length gene sequence contained two intron sequences of 109 bp and 127 bp (the underlined region in SEQ ID NO.1). 4DS-2 Three pairs of primers were designed to identify the intron positions in the full-length gene sequence (Table 1). 4DS-2 The full-length gene sequence (SEQ ID NO.1) was cloned into three segments to obtain RLK 4DS-2 Fragment 1 (186-RLK 4DS-2 -F1 / 186-RLK 4DS-2 -R1), fragment 2 (186-RLK 4DS-2 -F2 / 186-RLK 4DS-2 -R2) and fragment 3 (186-RLK 4DS-2 -F3 / 186-RLK 4DS-2 -R3), and finally RLK 4DS-2 Segment 1, RLK 4DS-2 Fragment 2 and RLK 4DS-2 Fragment 3 is combined together to remove RLK 4DS-2 The intron sequence in the full-length gene sequence was obtained by RLK 4DS-2 The CDs sequence of the gene is shown in SEQ ID NO.2:

[0029] RLK 4DS-2 CDs sequence of gene (SEQ ID NO.2):

[0030]

[0031] RLK 4DS-2 Protein sequence of gene (SEQ ID NO.3):

[0032] MLRRKFFTQNRGQLLKQLVSHRADIAERMLISLEELQKATNNFNQARRLGGGGHGTVY

[0033] KGILSDLHVVAIKKSNIVVKREIDEFINEVAILSQINHRNIVKLRGCCLETEVPLLAYEFIS

[0034] NGTLSDHLHTEEPRSLTWKDKLRITTETCKALAYLHSAVSVPVIHRDIKPSNILLDDALT

[0035] AKVSDFGASRYIAMNQTGTTTAVQGTIGYLDPTYYYSRRLTESSDVYSFGVLLIELLTR

[0036] RKPSLYRSNEGVGLVMEFVALLAEGNLSEILDPPVVEEGGSKVEDVANLAVSCVKLRA

[0037] EERPTMRQVEMALEALQARKEHVMGDLIEETNGKKHAAPNCPSTSKRPKGSGASRCYS

[0038] QEEEVVLSATFPR

[0039] Example 3: PUbi-RLK 4DS-2 Vector Construction and Stable Genetic Transformation of Wheat

[0040] Utilize RLK 4DS-2 The CDs sequence of the gene (SEQ ID NO.2) was used as a template to add homologous arms on both sides of the DNA by PCR, and then recombined with the plant expression vector PC186 that had been digested with BsrGⅠ by homologous recombination to obtain RLK 4DS-2 Gene overexpression vector PUbi::RLK 4DS-2 (like Figure 1The vector PC186 contains the PUbi promoter and the NOST terminator (Hao QQ, Wang WQ, Han XL, Wu JZ, Lyu B, Chen FJ, Caplan A, FuDL. (2018) Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum in barley. Molecular Plant Pathology 19: 1995-2010). After the vector construction was completed, wheat was stably transformed by Agrobacterium-mediated genetic transformation (Wang K, Shi L, Liang XN, Zhao P, Wang WX, Liu JX, Chang YN, Hiei Y, Yanagihara C, Du LP, Ishida YJ, Ye XG. (2022) The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nature Plants 8: 110-117), and transgenic wheat was positively screened (primer sequences are shown in Table 1; positive screening results are shown in the attached figure of the specification) Figure 2 The transgenic positive wheat plants showed a phenotype of thinning leaves and stems (as shown in the attached figure of the specification Figure 3 The statistical results showed that compared with the transgenic negative control, the width of the flag leaf of the positive wheat plant was reduced by 126%, the length did not change significantly, and the stem diameter was reduced by 98% (as shown in Table 2). In summary, the results in Example 3 prove that the wheat RLK cloned by the present invention 4DS-2 The gene has the function of regulating the thickness of wheat stems and leaves.

[0041] Table 2 PC186-RLK 4DS-2 Phenotypic data of transgenic positive plants

[0042]

[0043] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A gene regulating the thickness of wheat flag leaves and stems, characterized in that: The gene is RLK 4DS-2 , the RLK 4DS-2 The full-length nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The gene for regulating the thickness of wheat flag leaves and stems according to claim 1, characterized in that: The RLK 4DS-2 The CDs nucleotide sequence of the gene is shown in SEQ ID NO.2; the RLK 4DS-2 The amino acid sequence of the gene is shown in SEQ ID NO.

3.

3. The gene for regulating the thickness of wheat flag leaves and stems according to claim 1, characterized in that: The gene is from a 39 kb segment on Aegilops tauschii 4DS.

4. Use of the gene for regulating the thickness of wheat flag leaves and stems according to claim 1 in any one of the following (1) to (3): (1) Regulate the thickness of wheat flag leaves and stems; (2) Molecular marker breeding of wheat crops; (3) Morphological markers in the hybrid breeding system of wheat crops.

5. The use according to claim 4, characterized in that: For RLK 4DS-2 Gene overexpression vector PC186-RLK 4DS-2 , wheat was stably transformed by Agrobacterium-mediated genetic transformation, and transgenic positive wheat plants showed a phenotype of thinner leaves and stems.

6. The use according to claim 5, characterized in that: The thinning phenotype of leaves and stems of transgenic positive wheat plants can be directly observed with the naked eye in the adult stage without the need for molecular marker screening; using RLK 4DS-2 Genes establish morphological markers to assist in molecular marker breeding and hybrid breeding systems for wheat crops.

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