Application of rubber tree HbCPT8 gene in promoting plant growth and increasing glue yield

Abstract

The invention discloses application of a hevea brasiliensis HbCPT8 gene in promoting plant growth and increasing gum yield, and belongs to the field of genetic engineering.The hevea brasiliensis HbCPT8 gene is transferred into kok-saghyz through a plant expression vector to obtain a transgenic kok-saghyz positive plant, and it is found through comparison of the phenotype of the transgenic kok-saghyz and the gum content of the root of the transgenic kok-saghyz that the hevea brasiliensis HbCPT8 gene can be used for promoting plant growth and increasing gum yield. Compared with wild type kok-saghyz, the biomass and the rubber yield of the transgenic kok-saghyz are remarkably improved, which shows that the HbCPT8 gene plays an important role in regulating and controlling rubber synthesis and has important significance in cultivating rubber plant varieties capable of increasing the rubber yield.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to application of a rubber tree HbCPT8 gene in improving plant growth and rubber production. Background Art

[0002] Natural rubber (cis-polyisoprene) is an important industrial raw material and strategic material related to the national economy and people's livelihood. Although there are more than 2,000 rubber-producing plants in the world, the Brazilian rubber tree (Hevea brasiliensis) is currently the only source of almost all commercial natural rubber in the world. The biosynthesis of natural rubber is an extremely complex biological process, involving the participation of multiple proteins and enzymes. The comprehensive regulation of multiple internal and external factors ultimately promotes the efficient synthesis of rubber polymers. In the latex of the Brazilian rubber tree, cis-prenyltransferase (CPT) plays an important role in the biosynthesis of natural rubber. It catalyzes the polymerization of IPP monomers into long-chain natural rubber molecules, affecting the properties and yield of natural rubber. In the genome of the rubber tree, HbCPT6, HbCPT7 and HbCPT8 are three CPT genes highly expressed in the latex.

[0003] The roots of rubber grass (Taraxacum kok-saghyz Rodin) are rich in natural rubber. Its rubber production structure, rubber biosynthesis pathway, and natural rubber structure and properties are similar to those of the Brazilian rubber tree, and have potential application value. In addition, due to its short growth period, simple tissue culture, and relatively easy genetic transformation, rubber grass has become an ideal model plant for studying the development of latex tubes and the regulation of rubber biosynthesis in rubber-producing plants. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for increasing the biomass and rubber production of rubber grass.

[0005] The technical scheme of the present invention is: application of the rubber tree HbCPT8 gene in promoting plant growth and increasing rubber production, wherein the nucleotide sequence of the rubber tree HbCPT8 gene is as shown in SEQ ID No.1 or a degenerate sequence encoding the same protein as the nucleotide sequence shown in SEQ ID No.1.

[0006] Furthermore, the application method is: overexpressing the rubber tree HbCPT8 gene in plants, thereby increasing the biomass and rubber production of the plants.

[0007] Furthermore, the rubber output is rubber output.

[0008] Furthermore, the plant is a plant that can produce natural rubber.

[0009] Furthermore, the plant that can produce natural rubber is a rubber tree or rubber grass.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention transfers the rubber tree HbCPT8 gene into rubber grass through a plant expression vector to obtain a transgenic rubber grass positive plant, and compares the phenotype and root glue content of the transgenic rubber grass. It is found that in terms of phenotype, under greenhouse planting conditions, the transgenic rubber grass grows faster than the wild-type rubber grass, and the biomass is larger. When it grows to 5 months, the transgenic rubber grass has the phenomenon of premature bolting and flowering. After being transplanted into the soil and grown for six months, the root growth condition of the transgenic rubber grass is obviously better than that of the Xinjiang rubber grass, which explains to a certain extent that the expression of the HbCPT8 gene in rubber grass is conducive to the growth of the root of the rubber grass. Comparing the glue content of the wild type and the transgenic rubber grass, it is found that HbCPT8 can greatly improve the glue content of the root of the rubber grass. It can be seen that the HbCPT8 gene plays an important role in regulating rubber synthesis, which is of great significance for cultivating rubber plant varieties that can increase glue production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Comparison of the phenotypes of HbCPT8-transgenic rubber grass seedlings and Xinjiang rubber grass seedlings, left: transgenic rubber grass, right: Xinjiang rubber grass.

[0013] Figure 2 The phenotypes of HbCPT8-transgenic rubber grass seedlings and Xinjiang rubber grass seedlings were compared after being transplanted into the soil and grown for one month. Left: transgenic rubber grass, right: Xinjiang rubber grass.

[0014] Figure 3 Comparison of the root phenotypes of HbCPT8-transgenic rubber grass seedlings and Xinjiang rubber grass after they were transplanted into the soil and grown for 6 months. Left: Xinjiang rubber grass, right: transgenic rubber grass.

[0015] Figure 4 Comparison of rubber yield between HbCPT8 gene-transgenic rubber grass seedlings and Xinjiang rubber grass roots. DETAILED DESCRIPTION

[0016] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0017] Example 1: Cloning of the specific target fragment of the rubber tree HbCPT8 gene and construction of the expression vector

[0018] Based on the data collected from the Hevea brasiliensis, local blast and other bioinformatics tools were used to search, confirm and obtain the full-length gene sequence of the Hevea brasiliensis HbCPT8 gene cDNA (SEQ ID No. 1).

[0019] After determining the sequence information of the rubber tree HbCPT8 gene, specific primers are designed by Primer software for PCR amplification of the HbCPT8 gene. After the design is completed, the primers are synthesized by a synthesis company (such as Shanghai Shenggong). According to the operating instructions of the plant total RNA extraction kit, total RNA is extracted from the rubber tree and reverse transcribed to obtain cDNA. Using the designed specific primers, the cDNA of the rubber tree is PCR amplified. After the PCR reaction is completed, the product is subjected to agarose gel electrophoresis. If the resulting band is consistent with the expected HbCPT8 gene fragment size, the next step of the experiment can be carried out. The gel recovery of the target fragment is performed according to the instructions of the agarose gel DNA recovery kit. The recovered HbCPT8 gene fragment is cloned into the T-vector vector. After successful connection, it is sent to a sequencing company for sequencing to verify whether the inserted DNA fragment is the correct HbCPT8 gene sequence.

[0020] The bacterial liquid of the cloned strain that has been sequenced correctly is streaked on an LB plate containing the corresponding antibiotics, and colony PCR is performed after overnight culture. The positive clone is inoculated into 6-8 ml of liquid LB medium containing the corresponding antibiotics, and is cultured overnight at 37°C and 200 rpm in a shaking incubator for plasmid extraction. The plasmid extraction method is carried out according to the instructions of the plasmid mini-extraction kit, including cell lysis, DNA binding, washing, elution and other steps to purify the plasmid DNA.

[0021] Double enzyme digestion and overexpression vector construction, use Smal and Xbal double enzyme digestion to obtain the Super1300 plant expression vector large fragment and target fragment. After double enzyme digestion, agarose gel electrophoresis is performed to separate DNA fragments of different sizes. The target small fragment and the Super1300 plant expression vector large fragment are recovered, and the recovered HbCPT8 gene fragment is connected with the cut Super1300 plant expression vector fragment using T4 ligase to form a recombinant plasmid. The recombinant plasmid and the expression vector are then connected and sent to Shanghai Shenggong Bioengineering Company for sequencing. After success, the recombinant expression vector is transformed into DH5a Escherichia coli for bacterial transformation and recombinant plasmid amplification.

[0022] Positive clones were screened by colony PCR detection to ensure that the HbCPT8 gene had been successfully integrated into the expression vector. For clones that were positive in PCR detection, the recombinant plasmid was extracted and double-enzyme digestion was performed. The double-enzyme digestion results were detected by agarose gel electrophoresis to confirm that the size of the HbCPT8 target gene was consistent with the size of the cloned gene, thereby verifying that the plant expression vector was successfully connected. After confirming that the connection was successful, the recombinant expression vector can be used in subsequent transformation experiments to study the expression of the HbCPT8 gene in plants and its effect on rubber production.

[0023] Example 2: Acquisition of overexpressing rubber grass plants

[0024] In order to further understand the function of HbCPT8, the inventors conducted transgenic verification of HbCPT8. The specific process is briefly described as follows.

[0025] First, the constructed HbCPT8 expression vector was transferred into Escherichia coli for resistance screening, and positive colonies were selected for PCR detection. The colonies identified correctly by PCR were amplified and sent for sequencing. The plasmid was extracted from the bacterial solution with correct sequencing for later use.

[0026] Second, the extracted plasmid was transformed into Agrobacterium competent cells GV3101 and stored at 80°C for future use.

[0027] Third, select young and tender leaves of rubber grass, place them in the induction medium, and transfer them to the rooting medium after germination to obtain sterile seedlings. Select sterile seedlings with good growth, place them on sterile filter paper, cut off the leaf edges, cut the veins in the leaf disc, and cut the stems into about 1 cm in length, place them on the pre-culture medium, and culture them in the dark for 2 to 3 days.

[0028] Fourth, wild-type rubber grass (WT) was transformed by Agrobacterium infection. Agrobacterium containing the overexpression vector was streaked in an LB plate and inverted for 2 days. After the expanded culture, the MS impregnation solution was used to resuspend the thalline. The scratched leaves of the pre-culture were soaked at room temperature with the resuspended bacterial solution, then spread on sterile filter paper to dry the excess bacterial solution, transferred to the MS co-cultivation medium, and dark cultured for 2-3 days. The explants were taken out and transferred to the screening medium. According to their growth conditions, the resistant explants were transferred to the new screening medium every two weeks until resistant buds were grown, and then transplanted to the nutrient soil to obtain transgenic plants.

[0029] Fourth, transgenic rubber grass plants were obtained, and after the plants had induced root elongation for about 30 days, transgenic positive plants were detected. After three tests, the transgenic rubber grass positive plants were determined to be T0.

[0030] A total of 81 T1 generation rubber grasses (including Xinjiang rubber grass and transgenic HbCPT8 gene rubber grass) were planted, and the PCR results and the results of seed collection were statistically shown in Table 1.

[0031] Table 1 Statistics of T1 generation rubber grass materials

[0032]

[0033] The seeds of T1 HbCPT8 gene-transformed rubber grass materials with positive PCR test results were harvested to obtain T2 generation seeds.

[0034] Example 3: Verification of the function of the HbCPT8 gene in rubber grass

[0035] In order to explore the effect of the HbCPT8 gene on rubber plants and yield, the inventors compared the phenotypes and rubber content of transgenic plants and wild-type plants.

[0036] 1. Phenotypic comparison between transgenic plants and wild-type plants

[0037] Xinjiang rubber grass and the T2 HbCPT8 gene-transfected rubber grass seeds obtained in Example 2 were planted and hardened at the same time, and cultured under the same environmental conditions. After a period of time, they were compared and photographed, and the results were as follows:

[0038] The sterile seedlings grew well and were not contaminated. The leaves of Xinjiang rubber grass seedlings grew better than those of transgenic rubber grass seedlings, and the roots were longer, but the roots of transgenic rubber grass seedlings were thicker than those of Xinjiang rubber grass seedlings ( Figure 1 ). It is possible that the growth of transgenic rubber grass seedlings is worse than that of Xinjiang rubber grass due to the effect of antibiotics.

[0039] After being transplanted into the soil and growing for one month, the transgenic rubber grass showed significantly better growth than the Xinjiang rubber grass ( Figure 2 ), which to some extent shows that the expression of HbCPT8 gene in rubber grass is beneficial to the growth of rubber grass, and it has certain advantages compared with Xinjiang rubber grass.

[0040] After six months of growth in the soil, the root growth of the transgenic rubber grass was significantly better than that of the Xinjiang rubber grass ( Figure 3 ), which to some extent shows that the expression of HbCPT8 gene in rubber grass is beneficial to the growth of rubber grass roots.

[0041] 2. Comparison of gum content between transgenic plants and wild-type plants

[0042] Rubber content was measured by Fourier infrared spectroscopy: After about 12 months, the root tissues of wild-type and transgenic rubber grass were removed, dried, and then dried in an oven at 65°C for more than 3 hours. The root tissue was ground into powder. Deionized water was added, vortexed thoroughly, and the floating matter was retained after centrifugation. Then, it was placed in a rotary centrifugal concentrator to evaporate the water, and acetone was added to wash until it was colorless and transparent. The supernatant was removed, placed in an oven to remove the acetone, and petroleum ether was added for full extraction. Then, it was placed in an infrared spectrometer for detection, and the target peak areas at 835 cm and 699 cm were recorded.

[0043] By observing and comparing the phenotypic changes of HbCPT8 transgenic plants and wild rubber grass, it was found that the growth phenotype of rubber grass changed significantly during the same period. The transgenic rubber grass plants were significantly larger than wild rubber grass, and the biomass was higher than the wild type. This shows that after the transgenic, these genes regulated the metabolic activities in the rubber grass, making the transgenic rubber grass grow vigorously. The rubber content of the transgenic roots was determined using a Fourier infrared spectrometer. The results of the rubber content determination of rubber plants are as follows: Figure 4 As shown: the maximum value of the wild-type plant was 2.74%, the minimum value was 1.97%, and the average value was 2.37%; the maximum value of the transgenic plant was 7.15%, the minimum value was 3.95%, and the average value was 4.93%. Its rubber content is higher than that of the wild-type rubber grass. This result verifies the role of HbCPT8 in promoting rubber production. The expression of some protein isomers in the CPT protein family in rubber particles can balance the osmotic pressure inside and outside the cells, thereby promoting the rapid increase of latex production. The activity of CPT is one of the important factors affecting rubber content.

Claims

1. Application of the rubber tree HbCPT8 gene in promoting plant growth and increasing rubber production, wherein the nucleotide sequence of the rubber tree HbCPT8 gene is as shown in SEQ ID No.1 or a degenerate sequence encoding the same protein as the nucleotide sequence shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The application method is: over-expressing the rubber tree HbCPT8 gene in plants, thereby increasing the biomass and rubber yield of the plants.

3. The use according to claim 1 or 2, characterized in that: The rubber output is the rubber output.

4. The use according to claim 1 or 2, characterized in that: The plant is a plant that can produce natural rubber.

5. The use according to claim 4, characterized in that: The plant capable of producing natural rubber is a rubber tree or rubber grass.

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