Catalpa bungei RAP2-2 gene capable of promoting fast growth and stem abundance of woody plants and application thereof

Abstract

The invention relates to the field of plant molecular biology, in particular to a catalpa bungei CbuRAP2-2 gene capable of promoting fast growth and stem abundance of woody plants, a protein coded by the catalpa bungei CbuRAP2-2 gene and application. The gene for coding the catalpa bungei CbuRAP2-2 protein is obtained through cloning, and the sequence of the gene is as shown in SEQ ID No. 1. The gene is transferred into a poplar genome and is over-expressed in a transgenic poplar, so that the stem diameter of the poplar is increased. Therefore, the gene is beneficial to shortening the breeding period and rotation period of the woody plants, and has an excellent application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for promoting rapid growth and abundant fruit production in woody plants. CbuRAP2-2 Genes and their applications. Background Technology

[0002] Catalpa ( Catalpa bungei As a deciduous tree belonging to the genus *Catalpa* in the family Bignoniaceae, it is an important and valuable broad-leaved timber species and a well-known ornamental tree in central my country, with a long history of cultivation. Due to the long growth cycle and complex genome of woody plants, the efficiency of forest tree breeding is generally low, and the rotation period can last at least several years. Currently, forest tree genetics and breeding in my country is developing towards intelligent molecular design breeding, urgently requiring the compilation and establishment of gene banks influencing important traits of forest trees and the development of corresponding high-quality new germplasm.

[0003] RAP2.2 Current research on homologous genes mainly focuses on responses to abiotic stresses, particularly in Arabidopsis thaliana. RAP2.2 The induced expression of this compound conferred tolerance to hypoxia, oxidation, and osmotic stress in plants and enhanced their sensitivity to abscisic acid (ABA). Furthermore, RAP2.2 Homologous genes have also been reported to play important roles in plant growth and development. In the ethylene-mediated seed dormancy release process, RAP2.2 is located downstream of PRT6; ethylene reduces... RAP2.2 The expression of [something] promotes seed germination. Mingchao Qian et al. believe that European rapeseed ([something]) [is used to promote seed germination]. Brassica napus In ) BnaA05.RAP2.2 It may play a key role in the negative regulation of petal size through the ethylene signaling pathway. However, RAP2.2 The role of homologous genes in promoting the vegetative growth of woody plants remains unclear. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for promoting rapid growth and abundant trunk production in catalpa trees. CbuRAP2-2 Genes and their applications.

[0005] First, the present invention provides Catalpa CbuRAP2-2 protein, which is a protein composed of the amino acids shown in SEQ ID No. 2.

[0006] Secondly, the present invention also provides a gene encoding the Catalpa CbuRAP2-2 protein.

[0007] Preferably, the sequence of the gene is shown in SEQ ID No. 1.

[0008] The present invention also provides an overexpression vector containing the gene, a host cell, and an engineered bacterium.

[0009] The present invention also provides the use of the gene in promoting plant height growth and / or stem thickening.

[0010] In one specific embodiment of the present invention, the gene is transferred into the plant genome and the gene is overexpressed in the transgenic plant.

[0011] In one specific embodiment of the present invention, the plant is a woody plant, preferably a catalpa or poplar.

[0012] This invention obtains catalpa trees through homologous cloning. CbuRAP2-2 Genes. By constructing an overexpression vector and using Agrobacterium-mediated expression, the gene from Catalpa tree was expressed. CbuRAP2-2 The gene was integrated into the poplar genome. By comparing the phenotypes of transgenic and non-transgenic poplars, it was found that the gene overexpressed in *Cathaya argyrophylla* was... CbuRAP2-2 Genes can promote plant height and significantly thicken the stems of transgenic plant materials. Attached Figure Description

[0013] Figure 1 As shown CbuRAP2-2 Dynamic changes in gene expression levels in the xylem of Catalpa trees at different developmental stages.

[0014] Figure 2 The image shows the comparison of homologous amino acids in RAP2.2 between poplar and catalpa trees.

[0015] Figure 3 The image shows the identification of positive seedlings.

[0016] Figure 4 The image shows overexpression. CbuRAP2-2 It can effectively promote the synergistic increase of height and diameter growth in poplar trees. Detailed Implementation

[0017] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions or conditions recommended in the manufacturer's instructions. Example 1: RNA-seq identifies the dendrites of catalpa trees during their growth period.

[0018] Xylem samples were collected from four-year-old Catalpa trees in April, May, June, August, and September. RNA was extracted from the samples and subjected to transcriptome sequencing. TopHat v2.0.9 was used to align clean reads with the Catalpa reference genome. Cufflinks v2.1.1 was used to standardize and quantify transcript expression levels to calculate fragments per kilobase of transcripts per million mapped reads (FPKM). GraphPad Prism 9 was used for visualization analysis. The results showed that with spring budding, vigorous growth in summer, and slowing growth in autumn, the temperature gradually increased from April to August. CbuRAP2- 2 Gene expression levels increased synchronously. Figure 1 The analysis results suggest that... CbuRAP2-2 Genes are involved in the growth and development of catalpa trees.

[0019] Example 2 Creation CbuRAP2-2 Genetically modified poplar 2.1 Preparation of products containing CbuRAP2-2 Agrobacterium gene Using *Catalpa bungei* terminal bud cDNA as a template, cloned using primers CbuRAP2-2_F and CbuRAP2-2_R. CbuRAP2-2 Gene. An amino acid comparison between the isolated CbuRAP2-2 and poplar RAP2.2 (Potri.001G163700.5.v4.1) revealed only 52.50% amino acid sequence similarity. Figure 2 ),illustrate RAP2.2 Homologous genes are not conserved between two species.

[0020] Using primers OE-CbuRAP2-2_F and OE-CbuRAP2-2_R, CbuRAP2-2 The complete CDS was ligated to pBI121 via homologous recombination and then transformed into E. coli TOP10 competent cells. Samples were sent for sequencing, and bacterial cultures with correct sequences were selected for preparation of glycerol culture, which was stored at -80°C for later use.

[0021] Table 1 PCR Primers

[0022] After activating the bacteria by streaking with glycerol, the culture was expanded, plasmids were extracted, and transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method. Single clones were selected, and bacterial culture was verified by PCR using vector primers. After gel electrophoresis, bacterial cultures with correct bands were added to glycerol and stored at -80℃ for later use. 2.2 Infection of poplar leaves

[0023] Activate glycerol bacteria and expand culture to OD. 600 The bacterial cells were centrifuged at 4000 rpm for 10 min to reach a molecular weight of 0.6-0.8. The obtained bacterial cells were resuspended in liquid MS medium (containing 5% sucrose and 200 μM acetylsuccinone) to prepare Agrobacterium infection suspension.

[0024] '84K' Yang ( P. alba ♀× P. glandulosa (♂), a superior clonal line of Populus tomentosa obtained through hybridization, has been widely planted. An efficient and rapid genetic transformation system has been established, making it a model tree species for verifying gene function research in woody plants. Young leaves from tissue-cultured seedlings of '84K' Populus tomentosa were punctured with a blade, then immersed in a suspension for 2 hours. After being blotted dry with sterile filter paper, the leaves were placed on MS solid medium (containing 0.5 mg / L 6-BA and 30 g / L sucrose) and co-cultured in the dark for 2 days. The leaves were then transferred to a medium containing Kansas... + On MS solid medium (containing 0.5 mg / L 6-BA, 0.004 mg / L TDZ and 30 g / L sucrose), after 1 cm buds grew from the wound, the buds were repeatedly screened three times. DNA was extracted from the leaves of the healthy buds, and positive seedlings were identified by PCR using upstream and downstream primers OE-CbuRAP2-2_F and OE-CbuRAP2-2_R. Figure 3 Positive seedlings were selected and transferred to 1 / 2 MS rooting medium (25 g / L sucrose and 300 mg / L Cef) for normal light cultivation. Healthy transgenic seedlings were then transferred to a greenhouse substrate for further cultivation. 2.3 Observation of positive seedling phenotypes

[0025] In order to explore CbuRAP2-2 Its role in woody plants, and its overexpression in '84K' poplar. CbuRAP2-2 Phenotypic observations were conducted to study... CbuRAP2-2 biological functions ( Figure 4 By measuring plant height, number of internodes, and basal diameter, it was found that compared with the wild type (WT), overexpression of [specific gene] was significantly reduced. CbuRAP2-2 The height, number of internodes, and basal diameter of the transgenic poplar trees were significantly increased. Figure 4 A, 4B). For further research... CbuRAP2-2 The effect of overexpression on apical meristem (SAM) development: We obtained WT and overexpression results. CbuRAP2-2 Longitudinal tissue sections of SAM from transgenic poplar trees. We found that... CbuRAP2-2 After overexpression, the area of ​​poplar SAM increased significantly, and the basal diameter and height of SAM also increased significantly. P <0.05)( Figure 4 C, 4D). These results indicate that CbuRAP2-2Overexpression of SAM led to accelerated development of the base diameter and height of SAM, thereby promoting an increase in the overall development rate of transgenic poplar.

[0026] In addition, in order to conduct further research CbuRAP2-2 The effect of overexpression on wood development: We obtained WT and overexpression... CbuRAP2-2 Transverse sections of tissue from the 8th internode of transgenic poplar trees. We found that, compared with WT, overexpression... CbuRAP2-2 The xylem width of the transgenic poplar was significantly increased. Figure 4 E, 4F). Anatomical analysis showed that, CbuRAP2-2 Overexpression of [a specific substance] enhanced radial development of the xylem. These results indicate that... CbuRAP2-2 Overexpression of this substance leads to accelerated stem elongation and an increase in the number of internodes, resulting in a tall and thick-stemmed poplar phenotype.

[0027] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Catalpa CbuRAP2-2 protein, which is a protein composed of the amino acids shown in SEQ ID No.

2.

2. The gene encoding the Catalpa CbuRAP2-2 protein as described in claim 1.

3. The gene as described in claim 2, characterized in that, The sequence is shown in SEQ ID No.

1.

4. A vector containing the gene of claim 2 or 3.

5. Engineered bacteria containing the gene described in claim 2 or 3.

6. Use of the gene of claim 2 or 3 in promoting plant height growth and / or diameter increase.

7. The use as described in claim 6, characterized in that, The gene is transferred into the plant genome and overexpressed in the transgenic plant.

8. A method for promoting the growth of plant height and / or diameter of transgenic plants, characterized in that, Using Agrobacterium-mediated transformation, an overexpression vector containing the gene described in claim 2 or 3 is transferred into the plant genome, and transgenic plants are obtained through screening.

9. The method as described in claim 8, characterized in that, The gene was overexpressed in the transgenic plant.

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