Use of tea tree csbes gene

Abstract

The application belongs to the field of biotechnology, and particularly relates to the use of a tea tree CsBES gene for regulating leaf drooping of the tea tree. The application discloses application of the CsBES gene in regulating leaf shape of the tea tree, and the CsBES gene positively regulates leaf drooping. The tea tree obtained by silencing the CsBES gene has high leaf curvature and low leaf drooping degree.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology; specifically, it relates to the use of the CsBES gene in tea trees to regulate the drooping of tea leaves. Background Technology

[0002] Tea is one of the world's three major non-alcoholic beverages. my country boasts abundant tea germplasm resources and a large-scale tea industry with high output value. In the past, tea harvesting relied primarily on manual picking. Now, due to a shrinking workforce caused by an aging population, mechanized harvesting has gradually become a focus of the tea industry. Tea trees with traits suitable for mechanized harvesting have become a key focus of the tea industry's development. Leaf drooping is a major factor affecting the integrity rate of buds and leaves during mechanized harvesting. Therefore, how to regulate leaf drooping in tea trees has become a key focus in the breeding of tea trees suitable for mechanized harvesting. Plant hormones are natural and harmless; utilizing plant hormone response genes to regulate leaf drooping in mechanized tea trees provides a possible technical route to improve their suitability for mechanized harvesting.

[0003] The CsBES gene belongs to the bHLH class of transcription factors and is a component of the brassinosteroid (BR) signal transduction pathway. Previous studies have focused on the function of its homolog BES1 in regulating cell elongation and plant stress resistance in Arabidopsis thaliana, but the function of its tea homolog CsBES (CSS0038858) has not been reported.

[0004] The currently known gene involved in regulating leaf drooping in plants is the sage LRR receptor-like kinase gene DPY1. This gene and the CsBES gene belong to completely different gene families and originate from completely different species (tea tree is a dicotyledonous woody plant, while sage is a monocotyledonous herbaceous plant). Their nucleotide and amino acid sequences are also completely different, so there is no correlation between the two.

[0005] The sequence of the CsBES gene has been published in TeaGVD (http: / / www.teaplant.top / teagvd). No research reports have been published on this tea plant gene, and its uses are unknown. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide an application of the CsBES gene in regulating the degree of drooping of tea leaves.

[0007] To address the aforementioned technical problems, this invention provides the use of the CsBES gene for positively regulating leaf drooping. The nucleotide sequence of the CsBES gene is as described in SEQ ID NO: 1.

[0008] The amino acid sequence of the protein encoded by the CsBES gene is shown in SEQ ID NO: 2.

[0009] As an improvement to the use of the CsBES gene in this invention: the leaf is a tea leaf.

[0010] The CsBES gene is used to antisense suppress tea leaf drooping, which inhibits leaf drooping. Specifically, tea plants with silenced CsBES genes exhibit increased leaf curvature and reduced leaf drooping.

[0011] The antisense primers used for silencing the CsBES gene are as follows:

[0012] CsBES antisense primer-1: 5'-CTCTCTCCTCTTGTTGTTCT-3'

[0013] CsBES antisense primer-2: 5'-TTTGATTTGGGGGAGGTCTG-3'

[0014] CsBES antisense primer-3: 5'-GAAAGGCAGAATGTAAGATG-3'

[0015] CsBES antisense primer-4: 5'-TGGCCGGTGTAAGATGGTGG-3'

[0016] CsBES antisense primer-5: 5'-ATTTTGGAGGGAGCTCTGCT-3'.

[0017] This invention also provides a method for increasing the curvature of tea leaves by silencing the CsBES gene.

[0018] As an improvement to the above method: equal amounts of the above CsBES antisense primer-1, CsBES antisense primer-2, CsBES antisense primer-3, CsBES antisense primer-4, and CsBES antisense primer-5 are mixed and diluted with distilled water to prepare an antisense inhibition solution, wherein the concentration of each primer in the antisense inhibition solution is 20±2uM.

[0019] This invention is the first to construct a tea plant with a silenced antisense repressor gene of the CsBES gene and conduct functional studies. By measuring leaf curvature, it was found that the CsBES gene in tea plays a positive promoting role in leaf spread and drooping. That is, it causes the tea leaves to droop more towards a more curved shape. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Figure 1 It is a vector map used for sequencing the full-length coding region of the CsBES gene.

[0022] Figure 2 It is the expression level of the CsBES gene in tea leaves that are silenced by the antisense repressor gene of the CsBES gene.

[0023] Figure 3 The degree of leaf drooping curvature of tea leaves is determined by the silencing of the CsBES gene antisense repressor gene.

[0024] In the above figure:

[0025] Blank represents the blank control group, 28nBL represents the induction group, AS+28nBL represents the antisense inhibition group, and S+28nBL represents the negative control group. Different letters indicate significant differences between different groups (p<0.05). Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0027] I. Obtaining the full-length sequence of the CsBES gene from tea plants:

[0028] Primers for whole-genome amplification were designed using Primer Premier 6.0. The cDNA of one bud and one leaf of the tea cultivar Longjing 43, grown at the Tea Research Institute of the Chinese Academy of Agricultural Sciences, was used as a template (the cDNA extraction method was conventional, for example, CN104561025A). Specific primers CsBES-F and CsBES-R were designed, and the CsBES fragment was amplified by KOD-plus-NEO high-fidelity enzyme PCR.

[0029] The primer sequence is: CsBES-F: 5'-ATGACTGCCGGCGGATC-3'

[0030] CsBES-R: 5'-TTAAGTGTGGGCTTTTCCAC-3'

[0031] PCR amplification reaction system: 5 μL 10xPCR Buffer, 5 μL 2mM dNTP, 3 μL 25mM MgSO4, 1 μL KOD-plus-NEO, 32 μL ddH2O, 1 μL cDNA, 1.5 μL each of forward and reverse primers, totaling 50 μL. The PCR reaction program was: 94℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 53℃ annealing for 30 seconds, 68℃ extension for 2 minutes, 35 cycles; final extension at 68℃ for 5 minutes. The obtained PCR products were identified by 1% agarose gel electrophoresis. The amplified bands were then purified using an Axygen DNA gel electrophoresis kit. The purified products were constructed into the pEASY-Blunt-Zero vector (vector diagram shown). Figure 1 The recombinant plasmid was sent to Youkang Company for sequencing confirmation.

[0032] The nucleotide sequence of the obtained gene CsBES is shown in SEQ ID No: 1; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID No: 2.

[0033] II. Design and Synthesis of CsBES Gene Antisense Repression Primers and Negative Control Sensitive Primers

[0034] Using the Solido online design software, the obtained SEQ ID No: 1 nucleotide sequence was submitted. Based on the binding energy values ​​calculated by the software according to the complementary base pairing of nucleotides, five 20bp oligonucleotide regions were obtained from the SEQ ID No: 1 nucleotide sequence. These five regions (starting from the start codon) are 50-69bp, 231-250bp, 413-432bp, 633-652bp, and 779-798bp. Oligonucleotide chains with lower binding energies were selected from each of the five regions. Based on these oligonucleotide chains, deoxynucleotide chains “CsBES antisense primers-1-5” were designed as antisense repression primers. The complementary base chains of the antisense repression primers were used as negative control positive primers. Primers with an OD value of 200 were synthesized at a company called Youkang. The specific primer sequences are shown in Table 1 (all primers are in the 5' to 3' direction).

[0035] Table 1. Nucleotide sequences of CsBES gene antisense inhibitor primers and negative control sense primers

[0036]

[0037] III. Construction and Detection of CSBES Gene Antisense Repressor Materials:

[0038] The five CsBES antisense primers listed in Table 1 were mixed in equal amounts and diluted with distilled water to prepare an antisense inhibition treatment solution containing 20 μM of each primer. The five CsBES positive primers were also mixed in equal amounts and diluted with distilled water to prepare a negative control treatment solution containing 20 μM of each positive primer. Both the antisense inhibition treatment group and the negative control treatment group also required the addition of 5 μM of the brassinolide analog 28-homobrassinolide to ensure that tea leaf drooping was promoted within a certain time period (i.e., the time during which antisense inhibition takes effect). Thus, the antisense inhibition treatment group contained 20 μM of antisense inhibition treatment solution and 5 μM of 28-homobrassinolide, while the negative control group contained 20 μM of negative control treatment solution and 5 μM of 28-homobrassinolide. Simultaneously, a blank control group containing only distilled water and no hormones or primers, and an induction group containing 5 μM of 28-homobrassinolide were also set up.

[0039] One bud and one leaf of the Jianghua bitter tea variety, grown at the National Germplasm Hangzhou Tea Tree Nursery Base within the Tea Research Institute of the Chinese Academy of Agricultural Sciences, were harvested. It was ensured that the harvested bud and leaf were healthy shoots from the current year's new growth, and that they were of uniform size and growth status. The harvested buds and leaves were randomly divided into four groups: a blank control group (Blank), an induction group (28nBL), an antisense inhibition treatment group (CsBES AS+28nBL), and a negative control group (CsBES S+28nBL), with at least ten biological replicates per group. The groups were placed in a light incubator at approximately 25℃ with a 16h / 8h light / dark cycle. Liquid was replenished daily to prevent the buds and leaves from drying out. IV. Study on the CsBES gene antisense inhibition of tea leaf curvature.

[0040] On the 5th day of treatment, the buds and leaves were frozen in liquid nitrogen, ground using a ball mill, and RNA was extracted according to the Tiangen Polysaccharide Polyphenol RNA Kit method. cDNA was then extracted via reverse transcription, and the expression level of the CsBES gene in each group was determined using quantitative real-time fluorescence. The results are as follows: Figure 2 According to Figure 2 This indicates that antisense inhibition treatment reduces CsBES gene expression. Treatment of the negative control group did not affect CsBES gene expression.

[0041] Photographs of buds and leaves were taken, and ImageJ was used to statistically represent the leaf drooping curvature. Leaf drooping curvature refers to the ratio of the distance between the proximal and distal ends of the leaf blade to the length of the midrib. The lower the leaf curvature, the more severe the drooping of the leaf, and the more curved the leaf.

[0042] The results are as follows Figure 3 According to Figure 3 It can be seen that during the process of 28nBL-induced leaf drooping in tea, the expression of the antisense-repressed silenced CsBES gene increased the leaf curvature and inhibited the degree of leaf drooping compared to the induction group. However, the leaf curvature of the negative control group (CsBES S+28nBL) and the induction group (28nBL) decreased and the leaves became more curved. This indicates that the CsBES gene reduces leaf curvature and positively promotes the degree of leaf drooping.

[0043] Note: This industry standard uses gene silencing to verify gene function on a single bud and leaf, as described by Zhao et al., 2019 and Chen et al., 2020. Specific reference information is as follows:

[0044] Mingyue Zhao,Na Zhang,Ting Gao,Jieyang Jin,Tingting Jing,JingmingWang,YiWu,Xiaochun Wan,Wifried Schwab and Chuankui Song.Sesquiterpeneglucosylation mediated by glucosyltransferase UGT91Q2 is involved in themodulation of cold stress tolerance in tea plants.New Phytologist(2020)226:362–372.

[0045] Yongxian Chen, Xiangyang Guo, Ting Gao, Na Zhang, Xiaochun Wan, WilfriedSchwab, and Chuankui Song. UGT74AF3 enzymes specifically catalyze theglucosylation of4-hydroxy-2,5-dimethylfuran-3(2H)-one, an important volatile compound in Camellia sinensis. Horticulture Research (2020) 7:25.

[0046] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Silence CsBES The application of gene-based reagents in regulating tea leaf morphology is characterized by: CsBES The gene was obtained by amplifying Longjing 43 using primers; the primers were: CsBES-F: 5'-ATGACTGCCCGGCGGATC-3' CsBES-R: 5'-TTAAGTGTGGGCTTTTCCAC-3'; CsBES Tea plants that have undergone gene silencing have more curved leaves and less drooping leaves; CsBES The antisense primers used for gene silencing are as follows: CsBES antisense primer-1: 5' -CTCTCTCCTCTTGTTGTTCT-3' CsBES antisense primer-2: 5'-TTTGATTTGGGGGAGGTCTG-3' CsBES antisense primer-3: 5'-GAAAGGCAGAATGTAAGATG-3' CsBES antisense primer-4: 5'-TGGCCGGTGTAAGATGGTGG-3' CsBES antisense primer-5: 5'-ATTTTGGAGGGAGCTCTGCT-3'.

2. A method for increasing the curvature of tea tree leaves, characterized in that: Make CsBES Gene silencing; Equal amounts of CsBES antisense primer-1, CsBES antisense primer-2, CsBES antisense primer-3, CsBES antisense primer-4, and CsBES antisense primer-5 as described in claim 1 are mixed and diluted with distilled water to prepare an antisense inhibition solution, wherein the concentration of each primer in the antisense inhibition solution is 20±2 μM.

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