Transcription factor CsKNAT1 and application thereof in regulation and control of development of tea trees

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a transcription factor CsKNAT1 and application thereof in regulation and control of development of tea trees. The nucleotide sequence of the gene is as shown in SEQ ID NO: 1, and the sequence of protein coded by the gene is as shown in SEQ ID NO: 2. Research finds that overexpression of the CsKNAT1 gene can influence the form of plant leaves: the edges of the leaves are promoted to have notches (asymmetric leaves are formed, resulting in increase of sawteeth at the edges of the leaves); the formation of compound leaves can be promoted; leaves on plant stems can grow in a rosette form, and secondary stems are bolted in the middles of rosette leaves; the research also finds that the overexpression of the CsKNAT1 gene can obviously influence the flowering phase and the flower form of the plant; the delay of the flowering phase is promoted; overexpression of the CsKNAT1 gene can further influence development of floral organs and formation of siliques, part of arabidopsis plants have phenotypes that corolla becomes small, stamens are exposed, petals gradually extend and are zigzag in a wavy shape, the length of mature siliques is remarkably shortened by 20%-36.1%, and the phenomenon that the fruits are aborted occurs.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the transcription factor CsKNAT1 and its application in regulating tea tree development. Background Technology

[0002] Plant genetic engineering, as a core field of modern biotechnology, improves crop traits by regulating the expression of key genes, and has become an important means to enhance agricultural productivity and adaptability.

[0003] Transcription factors, acting as switches for gene expression regulation, play a decisive role in plant growth and development. However, research in economic crops such as tea (Camellia sinensis) lags behind. As a globally important beverage crop, the leaf morphology and flowering time of tea directly affect its yield, quality, and harvesting cycle, but the functional validation and application potential of tea transcription factors have not been fully explored, and targeted gene resources are lacking.

[0004] This invention studies and proposes the transcription factor CsKNAT1 and its application in regulating tea tree development. It reveals that CsKNAT1 can be used to influence plant leaf morphology, promote the formation of compound leaves, and affect the flowering period and flower morphology, providing new ideas for the breeding of tea tree varieties with different leaf morphologies and flowering periods. Summary of the Invention

[0005] The purpose of this invention is to provide the transcription factor CsKNAT1 and its application in regulating tea plant development. It reveals that CsKNAT1 can affect plant leaf morphology, promote the formation of compound leaves, and influence the flowering period and flower morphology, providing new ideas for the breeding of tea plant varieties with different leaf morphologies and flowering periods.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of the CsKNAT1 gene or CsKNAT1 protein in regulating tea plant development, wherein the nucleotide sequence of the CsKNAT1 gene is shown in SEQ ID NO.1;

[0008] The amino acid sequence of the CsKNAT1 protein is shown in SEQ ID NO.2.

[0009] Furthermore, the application in regulating tea tree development includes at least one of the following applications:

[0010] 1) Regulating leaf morphology;

[0011] 2) Regulating compound leaf formation;

[0012] 3) Regulate the flowering period of plants;

[0013] 4) Regulate the flower morphology of plants.

[0014] Furthermore, regulating leaf morphology includes:

[0015] Overexpression of the CsKNAT1 gene or increase in CsKNAT1 protein content promotes the appearance of notches on the leaf margins.

[0016] Furthermore, the regulation of compound leaf formation includes:

[0017] Overexpression of the CsKNAT1 gene or increase in CsKNAT1 protein content promotes the formation of compound leaves.

[0018] Furthermore, regulating leaf morphology includes:

[0019] Overexpression of the CsKNAT1 gene or increase in CsKNAT1 protein content promotes the growth of leaves on the plant stem in a rosette shape, and secondary stems emerge from the center of the rosette leaves.

[0020] Furthermore, regulating the flowering period of plants includes:

[0021] Overexpression of the CsKNAT1 gene or increased CsKNAT1 protein content can delay flowering.

[0022] Furthermore, regulating plant flower morphology includes:

[0023] Overexpression of the CsKNAT1 gene or increased CsKNAT1 protein content induces at least one of the following flower morphologies:

[0024] 1) The corolla becomes smaller;

[0025] 2) Stamens exposed;

[0026] 3) The petals gradually elongate and curve in a wavy pattern;

[0027] 4) The length of mature siliques is shortened;

[0028] 5) Fruit abortion.

[0029] This invention has at least the following beneficial effects:

[0030] This invention has found that overexpression of the CsKNAT1 gene can affect the morphology of plant leaves: promote the appearance of notches on the leaf edges (forming asymmetrical leaves and increasing the serrations on the leaf edges); promote the formation of compound leaves; and cause the leaves on the plant stem to grow in a rosette shape, with secondary stems emerging from the center of the rosette leaves.

[0031] The study also found that overexpression of the CsKNAT1 gene can significantly affect the flowering period and flower morphology of plants: it can delay the flowering period; overexpression of the CsKNAT1 gene can also affect the development of floral organs and the formation of siliques. Some Arabidopsis plants showed a phenotype with smaller corollas, exposed stamens, and petals that gradually elongated into a wavy and zigzag shape. The length of mature siliques was significantly shortened by 20% to 36.1%, and the fruits were aborted.

[0032] Therefore, this invention provides a new approach for cultivating tea varieties with different leaf morphologies and flowering periods, and has significant potential for production applications. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 A clone of the CsKNAT1 gene;

[0035] Figure 2 Subcellular localization of CsKNAT1 protein;

[0036] Figure 3 The expression patterns of the CsKNAT1 gene in different tissues of tea plants are shown. Lowercase letters in the bar chart indicate significance (P < 0.05).

[0037] Figure 4 To investigate the effects of the CsKNAT1 gene on plant and leaf morphology in Arabidopsis thaliana. (A) Morphology and leaf phenotype of wild-type (WT) and transgenic (OE) plants at week 3. (B) Morphology and leaf phenotype of transgenic (OE) plants at week 4. (C) Some OE plants exhibit compound leaf phenotype; red arrows indicate compound leaves. (D) OE plants have rosette leaves on their stems; red arrows indicate rosette leaves, and yellow arrows indicate secondary stems.

[0038] Figure 5 The phenotypic effects of CsKNAT1 overexpression on bolting and silique development in Arabidopsis thaliana. (A) At day 30, the 35S::CsKNAT1 transgenic line showed delayed flowering compared to the wild type. (B-C) The corolla of the OE plants was smaller, the stamens were exposed, and the petals gradually elongated and became wavy. (D) Comparison of silique length. (E) Seed abortion in OE plants. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example 1: Cloning and Subcellular Localization Analysis of the CsKNAT1 Gene

[0041] Methods: Young leaves of Fuding Dabaicha (Camellia sinensis (L.) O.Kuntze cv. FudingDabaicha) were used for RNA extraction. Total RNA was extracted using the EASYspin Plant RNA Rapid Extraction Kit (Beijing Adley Biotechnology Co., Ltd.), and RNA quality and concentration were detected using an ultra-micro spectrophotometer. cDNA was generated by reverse transcription using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing TransGen Biotechnology Co., Ltd.). The CsKNAT1 gene was amplified by PCR using cDNA as a template. The PCR amplification system was as follows: PrimeSTAR Max DNA Polymerase 25 μL, forward and reverse primers (ATGGAGGATTATAGTCAGCTGAGT & TCATGGCCCCAATCGGTAAGGACC) 1 μL each, cDNA as template 1 μL, and sterile water 21 μL. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 53℃ annealing for 10 s, 72℃ extension for 1 min 15 s, 38 cycles, and a final extension at 72℃ for 5 min. The PCR products (Tiangen Biotech Co., Ltd.) were recovered using a gel extraction kit, ligated into the pMD18-T vector, and transformed into DH5α *E. coli*. After successful colony PCR verification, the colonies were sent to Shanghai Sangon Biotech for sequencing.

[0042] Among them, the cloned CsKNAT1 gene, such as Figure 1 The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1, with a length of 1137 bp; the amino acid sequence encoded by the CsKNAT1 gene is shown in SEQ ID NO.2, encoding 378 amino acids, with a molecular weight of 43.3 kDa and a theoretical isoelectric point of 6.07. The CsKNAT1 protein contains four conserved domains: KNOX1 (116–155 aa), KNOX2 (171–215 aa), ELK (259–280 aa), and HD (299–338 aa). The CsKNAT1 protein is located in the nucleus of tobacco mesophyll cells (see [link to relevant documentation]). Figure 2 ), which is a nuclear localized protein.

[0043] SEQ ID NO.1:

[0044]

[0045] SEQ ID NO.2:

[0046] MEDYSQLSDNTAPRASFLYGGPVLAPSASVYGRTNSGSNVSNHHQTQMGMNSFHLQSGGCYQSESQSHPIVKTESGTSQHVQKFHYPSIIRGHQTVHHQQDHNNQQGNENSNDVDSIKAKIVAHPQYSNLLEAYMDCQKVGAPPEVVARLTAVRQEFEARQRASATCRDGSKDPELDQFMEAYYDMLVK YREELTRPLQEAMEFMRRTEAQLNLLNNGPVRIFNSEEKCEGVGSSEEEDQDNSGGETELPEIDPRAEDRELKNHLLRKYSGYLSSLKQELSKKKKKGKLPKEARQKLLNWWELHYKWPYPSETEKVALAESTGLDQKQINNWFINQRKRHWKPSEDMQFVMMDGIHPQNAAFYMEGHYMGEGPYRLGP

[0047] Example 2: Tissue expression pattern of the CsKNAT1 gene

[0048] Methods: Flowers, roots, stems, and leaves of Fuding Dabaicha (Camellia sinensis (L.) O.Kuntze cv. FudingDabaicha) were collected. Total RNA was extracted and reverse transcribed into cDNA, which was then quantitatively analyzed using real-time quantitative PCR. Specific primers (AACTGTTTGGTGCCCTCGAAT & GGCGTGGTTAGCTGAGGAAGT) were designed using the NCBI Primer-BLAST online tool (nih.gov). The real-time quantitative PCR system consisted of: 10 μL of SoFast EvaGreen Supermix enzyme, 0.5 μL each of forward and reverse primers, 1 μL of cDNA, and 8 μL of sterile water. The program was as follows: 95 ℃ pre-denaturation for 30 s, 95 ℃ denaturation for 30 s, 60 ℃ for 30 s, 40 cycles; melting: starting from 65 ℃, the temperature was increased to 95 ℃ at a rate of 0.5 ℃ every 5 s. Using the tea plant CsACTIN gene as an internal reference gene, three biological replicates were set up, and two... -ΔΔCT The method calculates the relative expression level of genes.

[0049] See Figure 3 , Figure 3The expression patterns of the CsKNAT1 gene in different tissues of tea plants were shown (P < 0.05). The results showed that CsKNAT1 was significantly highly expressed in the stems and roots of tea plants, and its expression level was relatively high in young buds during leaf development.

[0050] Example 3: Effects of CsKNAT1 gene overexpression on leaf morphology and flowering time in Arabidopsis thaliana

[0051] Methods: The CDS sequence of CsKNAT1 was constructed downstream of the 35S strong promoter of the pBA-myc plant expression vector. After successful construction, it was transformed into Agrobacterium GV3101. Colony PCR was performed to identify positive clones, which were then encapsulated in a solution containing 25 mg / L of [a specific antibiotic]. -1 Gen+50mg.L -1 Spe + 20 mg.L -1 The bacteria were cultured overnight in LB broth containing Rif antibiotics at 28°C. The bacterial cells were collected and suspended in a solution of 5% sucrose + 0.05% Silwet-L77, with the OD600 adjusted to approximately 0.8. Newly flowering wild-type Arabidopsis thaliana (ecotype Columbia) was used for flower-dipping infection, soaking for 30 seconds. The infected Arabidopsis was then kept moist with plastic wrap, kept in the dark for 24 hours, and then cultured normally. The infection was repeated once after approximately one week. Seeds harvested from Arabidopsis thaliana infected with Agrobacterium tumefaciens were designated as generation T0. These T0 seeds were then seeded in MS medium supplemented with 0.01% Basta for selection of transgenic Arabidopsis. Seeds successfully inoculated with the gene germinated normally on the medium and developed green leaves, while the remaining seeds either failed to germinate or produced yellow seedlings. When the green seedlings reached the 4-leaf stage, they were transplanted from the medium to a soil substrate for further growth, and seeds were harvested individually from each plant; these were designated as generation T1 transgenic Arabidopsis seeds. This process was repeated until pure lines of generation T3 were obtained. These seeds were then preserved for subsequent experiments.

[0052] result:

[0053] (1) Overexpression of CsKNAT1 affects plant leaf morphology (see Figure 4 CsKNAT1 promotes notching at the leaf margins, resulting in asymmetrical leaves: In OE transgenic plants, a distinct notched leaf phenotype was observed, and this phenotype became increasingly pronounced with growth and development, with lobes even extending to the midrib to produce leaflets. Figure 4 China A Figure 4 (B) CsKNAT1 promotes compound leaf formation: 2.8% of the leaves in OE transgenic plants were compound leaves, including bilobed, trilobed, and multilobed leaves. Figure 4(C). In addition, some OE Arabidopsis plants have leaves growing in a rosette shape on their stems, and secondary stems emerge from the center of the rosette leaves. Figure 4 (D).

[0054] (2) Overexpression of CsKNAT1 affects the flowering period and flower morphology of plants (see Figure 5 Table 1): CsKNAT1 delayed flowering: Compared with wild-type plants, most OE plants had their flowering time delayed by about 5-10 days, and a small number of OE plants had their flowering time delayed by up to 2 months, or even did not flower at all. Figure 5 (See Table 1, A). CsKNAT1 affects floral organ development and silique formation: Most OE Arabidopsis plants exhibited normal floral development, while approximately 2% of OE Arabidopsis plants showed a smaller corolla, exposed stamens, and gradually elongated, wavy petals. Furthermore, the length of mature siliques was significantly shortened by 20%–36.1%, and fruit abortion was observed. Figure 5 (B~E, Table 1)

[0055] Table 1. Statistics on bolting time and fruit length of WT and OE Arabidopsis thaliana (n=40, p<0.05)

[0056]

[0057] Note: 1 Statistics on the time points when the main inflorescence stem reaches 5 mm in length

[0058] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. CsKNAT1 The application of the gene or CsKNAT1 protein in regulating tea plant development is characterized by, The CsKNAT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The amino acid sequence of the CsKNAT1 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The application in regulating tea tree development includes at least one of the following applications: 1) Regulating leaf morphology; 2) Regulating compound leaf formation; 3) Regulate the flowering period of plants; 4) Regulate the flower morphology of plants.

3. The application according to claim 2, characterized in that, in, Controlling leaf morphology includes: overexpression CsKNAT1 Genes may increase the content of CsKNAT1 protein, promoting the appearance of notches on the leaf edges.

4. The application according to claim 2, characterized in that, in, Regulating compound leaf formation includes: overexpression CsKNAT1 Genes may increase the content of CsKNAT1 protein, promoting the formation of compound leaves.

5. The application according to claim 2, characterized in that, in, Controlling leaf morphology includes: overexpression CsKNAT1 Genes or increased CsKNAT1 protein content promote the growth of leaves on the plant stem in a rosette shape, and secondary stems emerge from the center of the rosette leaves.

6. The application according to claim 2, characterized in that, in, Regulating the flowering period of plants includes: overexpression CsKNAT1 Genes may increase the content of CsKNAT1 protein, thus delaying the flowering period.

7. The application according to claim 2, characterized in that, in, Regulating plant flower morphology includes: overexpression CsKNAT1 Genes or increased CsKNAT1 protein content can induce at least one of the following flower morphologies: 1) The corolla becomes smaller; 2) Stamens exposed; 3) The petals gradually elongate and undulate in a wavy pattern; 4) The length of mature siliques is shortened; 5) Fruit abortion.

Images