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Application of cotton steroid C22alpha-hydroxylase gene GhCYP90B1 to improvement of tomato quality

A technology of α-hydroxylase and steroids, which is applied in the field of plant genetic engineering, can solve the problems of low physiological activity and achieve the effects of increased single fruit weight, increased soluble protein and Vc content, and larger fruit

Inactive Publication Date: 2015-03-11
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that the substrate of AtDWF4 accumulates in large quantities in plants but has very low physiological activity, while the direct product of AtDWF4 has very low content in plants but has high physiological activity

Method used

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  • Application of cotton steroid C22alpha-hydroxylase gene GhCYP90B1 to improvement of tomato quality
  • Application of cotton steroid C22alpha-hydroxylase gene GhCYP90B1 to improvement of tomato quality
  • Application of cotton steroid C22alpha-hydroxylase gene GhCYP90B1 to improvement of tomato quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The cloning of embodiment 1GhCYP90B1 gene sequence

[0043] 1. Extraction of Cotton RNA

[0044] Select about 3g of fresh cotton material, quickly grind it into a fine powder in liquid nitrogen, put it into a 50mL centrifuge tube, add 15ml of 65°C preheated RNA extraction solution (2% CTAB (W / V), 2% PVP (W / V ), 100mmol / L Tris-HCl (pH8.0), 0.5g / L Spermidine, 2.0mol / L NaCl, 2% mercaptoethanol (V / V, added before use)), and mix well by inversion. Water bath at 65°C for 3-10 minutes, during which time mix 2-3 times. Chloroform:isoamyl alcohol (24:1) extracted twice (10,000r / min, room temperature, 5min). Take the supernatant, add 1 / 4 volume of 10mol / L LiCl solution, place at 4°C for 6h, and extract once each with chloroform:isoamyl alcohol (25:24:1) (10,000r / min, room temperature, 5min). Add 2 times the volume of absolute ethanol, and precipitate in a -70°C refrigerator for more than 30 minutes. Centrifuge at 12,000r / min at 4°C for 20min, discard the supernatant. The pre...

Embodiment 2

[0064] Construction of embodiment 2 overexpression vector and genetic transformation of tomato

[0065] 1. Construction of overexpression vector

[0066] The pUC-GhCYP90B1 vector was constructed when cloning the GhCYP90B1 gene, and the GhCYP90B1 fragment on it has been sequenced. The plant expression vector is a transformed pCambia vector (i.e. the pCambia-35S-MCS-NOS-NPT II vector described below), the HPT II gene of the vector is cleaved with XhoI and replaced with the NPT II gene and amplified from the pBI121 vector The primers were designed with XhoI sites at both ends, and the primer sequences were: NPT-1: 5'-CCGCTCGAGCGGGGATCTGGATCGTTTCGCATG-3' (SEQ ID NO.10), NPT-2: 5'-CCGCTCGAGCGGCTCAGAAGAACTCGTCAAGAAG-3' (SEQ ID NO.11 ), restriction enzyme digestion and sequencing results verified the direction of NPT II gene. The CaMV35S promoter and NOS terminator were amplified in the pBI121 vector, and the corresponding restriction sites were also introduced at both ends of the ...

Embodiment 3

[0086] Embodiment 3 detects the expression level of GhCYP90B1 gene in transgenic tomato

[0087] According to the method for extracting cotton RNA in Example 1, the total RNA of transgenic tomato and wild-type tomato leaves was extracted, and a cDNA strand was synthesized. Real-Time PCR was used to analyze the expression of the target gene in the transgenic tomato. The PCR was carried out on a quantitative real-time PCR instrument, and 12.5 μL MIX buffer (including PCR buffer, DNA polymerase, dNTPs and MgCl2) was included in the 25 μL reaction system. , provided by quantitative real-time PCR kit, Bio-Rad). The GhCYP90B1 gene was amplified with primers D4-500 (SEQ ID NO.9) and GhCYP90B1-2 (SEQ ID NO.6). The internal standard was the tomato tubulin gene (LeTUB, DQ205342), and the primer was LeTUB-1 (SEQ ID NO.6). NO.7) and LeTUB-2 (SEQ ID NO.8). The amplification program is: pre-denaturation at 94°C for 3min; 94°C, 30sec; 56°C, 30sec; 72°C, 30sec; the preset cycle number is 35...

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Abstract

The invention discloses application of a cotton steroid C22alpha-hydroxylase gene GhCYP90B1 to improvement of tomato quality and belongs to the field of plant genetic engineering. The application is mainly implemented by preparing a transgenic tomato plant of a high-expression GhCYP90B1 gene. The expression of the GhCYP90B1 gene in tomatoes is controlled by utilizing a constitutive plant promoter CaMV 35S, growth of the tomato plant can be promoted, and germination of transgenic tomato seeds and growth of roots are promoted. Meanwhile, transgenic tomato fruits are enlarged, the weight of a single fruit is increased, and the content of soluble sugar, soluble protein and Vc in the transgenic tomato fruits is obviously increased. The GhCYP90B1 gene can be applied to improved quality breeding of tomato fruits.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to the application of a cotton steroid C22α-hydroxylase gene GhCYP90B1 in improving tomato quality. Background technique [0002] Brassinosteroids (BRs) are a kind of steroid plant hormones widely present in plants. BRs play an important role in many physiological processes of plant growth and development, and the concentration is extremely low (nmol L -1 ) BRs can show extremely high physiological activity, so brassinosteroids are considered to be the sixth largest plant hormones after auxin, gibberellin, cytokinin, abscisic acid and ethylene. [0003] Studies on the model plant Arabidopsis thaliana have shown that deletion mutants of BRs synthesis and signal transduction pathways exhibit extremely dwarf phenotypes, as well as phenotypes such as smaller leaves, fewer flowers, and extremely low seed setting rates. Overexpressing the BRs gene in Arabidopsis can ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/84A01H5/00
Inventor 罗明李芳叶树娥李先碧裴炎
Owner SOUTHWEST UNIVERSITY
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