A rubber tree HbSPL7 gene and its application

Abstract

The present invention provides a gene related to the development of laticifer, named as rubber tree HbSPL7 The present invention also provides a protein encoded by the gene. The present invention clones and obtains a rubber tree from a rubber tree. HbSPL7 Gene, studies have shown that this gene is related to the development of latex in rubber trees, and its expression is significantly upregulated by coronatine. Transforming this gene into dandelions can significantly increase the number of latex cells and latex in dandelions, which has important application prospects in the future transgenic improvement of the number of latex and natural rubber yield in rubber trees and dandelions.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and specifically relates to a rubber tree HbSPL7 Genes and their applications. Background Art

[0002] Hevea brasiliensis ( Hevea brasiliensis Muell. Arg., referred to as rubber tree) is a tree of the genus Hevea in the Euphorbiaceae family, native to the Amazon River basin in South America, and mainly distributed in tropical regions. More than 98% of the world's natural rubber comes mainly from rubber trees. Laticifers are the place where natural rubber is synthesized and stored. The number of laticifers in the trunk bark is significantly positively correlated with the natural rubber yield (Hao BZ, Wu J L. Biology of laticifers in Hevea and latexproduction. Chinese Journal Trop Crops, 2004, 25(4): 1-7). Therefore, the regulatory mechanism of latex development is a scientific problem that urgently needs to be solved in rubber tree biological breeding, and is crucial to increasing the yield of natural rubber. Summary of the invention

[0003] The object of the present invention is to overcome the deficiencies in the prior art and provide a rubber tree HbSPL7 Genes and their applications.

[0004] The first aspect of the present invention is to provide a gene related to the development of the laticifer, named as rubber tree HbSPL7 Gene, nucleotide sequence as SEQ ID NO:1.

[0005] The second aspect of the present invention is to provide a protein encoded by the gene of the first aspect of the present invention.

[0006] The third aspect of the present invention is to provide a recombinant vector containing the gene described in the first aspect of the present invention.

[0007] The original vector of the recombinant vector can be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The present invention is not limited to this. In a specific embodiment of the present invention, the original vector is a pB7WG2D vector, etc., but it should be understood that the present invention can also use other plasmids, or viruses, etc.

[0008] The fourth aspect of the present invention is to provide a host bacteria or a transgenic cell line or a recombinant bacteria containing the gene of the first aspect of the present invention.

[0009] The fifth aspect of the present invention is to provide the use of the gene described in the first aspect of the present invention, or the protein described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention, or the host bacteria or transgenic cell line or recombinant bacteria described in the fourth aspect of the present invention in increasing the number of plant laticifer cells and / or laticifers.

[0010] Preferably, the plant is dandelion.

[0011] Among them, the gene described in the first aspect of the present invention, or the protein described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention, or the host bacteria or transgenic cell line or recombinant bacteria described in the fourth aspect of the present invention increases the number of plant laticifer cells and / or laticifers but does not change the plant phenotype.

[0012] The sixth aspect of the present invention is to provide use of the gene described in the first aspect of the present invention or the protein described in the second aspect of the present invention in responding to coronatine.

[0013] Wherein, the gene described in the first aspect of the present invention or the protein described in the second aspect of the present invention is significantly up-regulated in response to coronatine induction.

[0014] The seventh aspect of the present invention is to provide a primer pair, wherein the primer pair is pB7WG2D- HbSPL7 -F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAAAATGACGGTAACTTGT-3' and pB7WG2D- HbSPL7 -R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAAACTACATGGGCTCCTGCC-3'; or the primer pair is HbSPL7 -F:5'-ATGGAAAATGACGGTAACTTGT-3', HbSPL7 -R: 5'-TTAAACTACATGGGCTCCTGCC-3'; or the primer pair is HbSPL7-qF :5'-CTATAGCGTCGTGGGTGGTC-3', HbSPL7 -qR: 5'-TTGGGAGCCTTGGAATGCAT -3'.

[0015] The present invention clones and obtains rubber tree from rubber tree HbSPL7Gene, studies have shown that this gene is related to the development of latex in rubber trees, and its expression is significantly upregulated by coronatine. Transforming this gene into dandelions can significantly increase the number of latex cells and latex in dandelions, which has important application prospects in the future transgenic improvement of the number of latex and natural rubber yield in rubber trees and dandelions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For rubber tree in embodiment 1 HbSPL7 Gene expression patterns in bark treated with coronatine (COR), HbSPL7 Gene expression is upregulated by COR.

[0017] Figure 2 Rubber tree HbSPL7 Genetic transformation of dandelion plants with resistance genes BAR and HbSPL7 PCR detection of genes.

[0018] Figure 3 For wild-type dandelion (WT) and overexpression HbSPL7 Genetic dandelion ( HbSPL7 -OE) HbSPL7 Gene fluorescence quantitative expression detection results.

[0019] Figure 4 For wild-type dandelion (WT) and overexpression HbSPL7 Genetic dandelion ( HbSPL7 -OE) Microscopic structure of the plant root. Red arrows indicate the latex cells. Scale bar = 250 μm.

[0020] Figure 5 For wild-type dandelion (WT) and overexpression HbSPL7 Genetic dandelion ( HbSPL7 -OE) Statistics of root laticifer cell index.

[0021] Figure 6 For wild-type dandelion (WT) and overexpression HbSPL7 Genes of dandelion plants. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0023] Embodiment 1: HbSPL7 Gene cloning

[0024] The inner bark of rubber tree Reyan 7-33-97 was used as the material, and the genomic DNA was extracted using the DNAsecure new plant genomic DNA extraction kit (DP320) of TIANGEN. The genomic DNA was used as the template and TaKaRa's PrimeSTAR® Max DNA Polymerase Ver.2 was used to isolate the genomic DNA of the rubber tree. HbSPL7 The gene was amplified by PCR. The forward primer used was pB7WG2D- HbSPL7 -F is: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAAAATGACGGTAACTTGT-3'; reverse primer pB7WG2D- HbSPL7 -R is: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAAACTACATGGGCTCCTGCC-3'. The PCR amplification system is 50ul: PrimeSTAR Max Ver.2 Premix (2X) 25 μL, forward and reverse primers (10 μM) 2.5 μL each, DNA template 2 μL, ddH2O 18 μL. The PCR amplification program is: 95 °C pre-denaturation for 2 min; 98 °C denaturation for 10 s, 60 °C annealing for 15 s, 68 °C extension for 20 s, a total of 30 cycles; 72 °C extension for 10 min.

[0025] The PCR amplification products were separated by 0.8% agarose gel electrophoresis and the target bands were recovered. The target bands were connected to the plant overexpression vector pB7WG2D by the Gateway method and transformed into Escherichia coli. The positive clones were selected for sequencing (Shanghai Bioengineering Co., Ltd.). HbSPL7 The coding region sequence of the gene is shown in SEQ ID No: 1, and the protein encoded by the gene is shown in SEQ ID No: 2.

[0026] Example 2: Rubber Tree HbSPL7 Expression analysis in bark treated with coronatine (COR)

[0027] The second elongation unit of the rubber tree Reyan 7-33-97 sprout was treated with 20 μM COR. The inner bark was collected after 1 hour (1 h), 2 h, 4 h, 8 h, 1 day (d), 2 d, and 3 d of treatment. The total RNA was extracted using TIANGEN's RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441). The control was water treatment. The RNA of 1 hour (1 h), 2 h, and 4 h of the early response to COR treatment was mixed and a library was constructed for transcriptome sequencing, named COR-A, and the control was Control-A. The RNA of 8 hours (8 h), 1 d, 2 d, and 3 d of the late response to COR treatment was mixed and a library was constructed for transcriptome sequencing, named COR-B, and the control was Control-B. Through transcriptome sequencing, the transcriptome annotation and differential analysis were performed using the Reyan 7-33-97 genome as the internal reference gene. The results showed that compared with the control, the RNA in the inner bark of the rubber tree sprout was 1.5 times that of the control. HbSPL7 The expression was upregulated by COR treatment (Fig. 1).

[0028] Embodiment 3: HbSPL7 Genetically modified dandelion

[0029] 1. Acquisition and identification of transgenic dandelions

[0030] The strain with the correct sequencing obtained in Example 1 was expanded and cultured, and plasmids were extracted according to the instructions of the plasmid DNA small-scale purification kit (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.). HbSPL7 The pB7WG2D plasmid containing the gene was transformed into Agrobacterium tumefaciens strain AGL1 to obtain pB7WG2D-35S:: HbSPL7 of Agrobacterium tumefaciens AGL1.

[0031] The pB7WG2D-35S:: HbSPL7 The root segments of dandelions were infected with Agrobacterium tumefaciens AGL1 for genetic transformation, and Basta resistance screening was used to obtain resistant seedlings. The Basta resistance genes BAR and HbSPL7 Gene screening of transgenic positive plants from resistant seedlings. Specific method: Extract genomic DNA from dandelion leaves, use BAR and HbSPL7 The primers of BAR gene were used for PCR amplification, and then 1% agarose gel electrophoresis was performed to identify BAR gene and HbSPL7 The lengths of the PCR products of the BAR gene and HbSPL7 If the PCR products of the genes all have specific bands, the plant is a transgenic plant (also called an overexpression plant). HbSPL7As a positive control, water and wild-type dandelion genomic DNA were used as negative controls. The electrophoresis images of some plants are shown in Figure 2 The primer sequences were BAR-F: 5′-GAAGTCCAGCTGCCAGAAAC -3′, BAR-R: 5′-CTACCATGAGCCCAGAACGA -3′; HbSPL7 -F:5'-ATGGAAAATGACGGTAACTTGT-3', HbSPL7 -R: 5'-TTAAACTACATGGGCTCCTGCC-3'.

[0032] 2. Transfer pB7WG2D-35S:: HbSPL7 Expression detection and phenotypic analysis of positive plants

[0033] Root segments of wild-type and transgenic positive plants were taken and total RNA was extracted according to the instructions of RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) of TIANGEN. RNA was reverse transcribed using RevertAid First Strand Cdna Synthesis Kit of thermoscientific. The reverse transcribed cDNA was used as a template and TB Green ® Premix Ex Taq™ II (Tli RNaseH Plus) was used for quantitative expression analysis. Reaction system: TB Green Premix Ex Taq II (2X) (Tli RNaseH Plus) 10μl, HbSPL7 -qF primer (10 μM) 0.4 μl, HbSPL7 -qR primer (10μM) 0.4μl, cDNA 1μl, add ddH2O to make up to 20μl. Reaction program: 95℃ 2min; 94℃ 10s, 60℃ 20s, 72℃ 30s, 45 cycles; melting curve analysis. Primer sequences are TkGAPDH-qF: 5'-AGTTGGTTTCGTGGTATGAC -3', TkGAPDH-qR: 5'-ACATGTCAGTGAACAGGTAGAC -3'; HbSPL7-qF :5'-CTATAGCGTCGTGGGTGGTC-3', HbSPL7 -qR: 5'-TTGGGAGCCTTGGAATGCAT -3'. TkGAPDH was used as the internal reference gene and 2 -ΔΔCt Counting roots of wild-type and transgenic plants HbSPL7The relative expression of genes. Fluorescence quantitative analysis showed that HbSPL7 Gene overexpression strains HbSPL7 -OE1, HbSPL7 -OE2, HbSPL7 -OE3 had a significantly upregulated expression level compared with the wild-type plants ( Figure 3 ).

[0034] The roots of the three transgenic lines and the wild type (WT) were sectioned in paraffin for root microstructure analysis. The specific method is to fix the cut root material in 80% ethanol for 24 hours, and refer to the display method of the latex of rubber tree (Tian Weimin, Shi Minjing, Tan Haiyan, Wu Jilin, Hao Bingzhong, "Structure and Development of Rubber Tree Bark", Beijing: Science Press), and then slice it through gradient dehydration, iodine-bromide staining, paraffin embedding, and then observe it under an optical microscope. The latex cell index was statistically analyzed using ImageJ software, which represents the proportion of the latex area to the entire root area. The results showed that the number of latex cells in the transgenic plants increased significantly (Figure 4). The statistical results showed that the average latex index of the wild type WT was 0.213, while the three transgenic lines HbSPL7 -OE1, HbSPL7 -OE2, HbSPL7 -OE3 average milk duct index were 0.513, 0.664, 0.492, all of which were significantly different from the control group (p < 0.05) ( Figure 5 ), while there was no significant difference in phenotype between transgenic plants and wild-type plants ( Figure 6 ).

[0035] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Overexpression of Hevea brasiliensis HbSPL7 Hevea brasiliensis HbSPL7 Application of gene recombinant vector in increasing the number of dandelion latex tube cells and / or latex tubes; the rubber tree HbSPL7 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Hevea brasiliensis HbSPL7 Hevea brasiliensis HbSPL7 The recombinant vector of the gene increases the number of dandelion laticifer cells and / or laticifers but does not change the dandelion phenotype.

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