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Protein cyp734a1 like‑1 specifically expressed in fibroblasts and its application

A technology of cyp734a1like-1 and ghcyp734a1l-1, applied in the field of protein CYP734A1like-1 and its application

Active Publication Date: 2018-04-20
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a new option for improving the quality of cotton fiber, so as to improve the specific or dominant expression of the currently obtained genes, there are still too few genes to meet the needs of molecular design for improving cotton fiber yield and quality.

Method used

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  • Protein cyp734a1 like‑1 specifically expressed in fibroblasts and its application
  • Protein cyp734a1 like‑1 specifically expressed in fibroblasts and its application
  • Protein cyp734a1 like‑1 specifically expressed in fibroblasts and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Cloning and sequence analysis of GhCYP734A1L-1 gene

[0060] 1. Extraction of Cotton RNA

[0061] 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) extraction 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 (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 supernatan...

Embodiment 2

[0085] Example 2 Expression analysis of GhCYP734A1L-1 gene in cotton plant and fiber development

[0086] Using the synthesized cDNA strand as a template, real-time quantitative PCR kit (Bio-Rad) was used for PCR. The expression primers were designed based on the cDNA sequence of the GhCYP734A1L-1 gene, the 5' end primer was GhCYP734A1L-1-1 (SEQ ID NO.6), and the 3' end primer was GhCYP734A1L-1-2 (SEQ ID NO.7). Include 10 μL of MIX buffer (including PCR buffer, DNA polymerase, dNTPs, and MgCl) in a 20 μL reaction system 2 ), 1 μL (5 μmol / L) each of the 5’-end and 3’-end primers. The cycle parameters are 94°C pre-denaturation for 3 minutes; 94°C, 30sec, 55°C, 30sec, 72°C, 30sec, and the preset cycle number is 40. Cotton GhHISTONE3 gene (GenBank accession number: AF024716) was used as internal standard, the 5'-primer of GhHISTONE3 gene was GhHIS-1 (SEQ ID NO.8), and the 3'-primer was GhHIS-2 (SEQ ID NO.9).

[0087] 1. Expression patterns of different tissues and organs and di...

Embodiment 3

[0092] Example 3 Construction of overexpression and antisense suppression GhCYP734A1L-1 gene plant expression vector and genetic transformation of cotton

[0093] 1. Construction of excess and antisense expression vectors

[0094] The pGEm-GhCYP734A1L-1 vector was constructed when cloning the GhCYP734A1L-1 gene, and the GhCYP734A1L-1 fragment on it has been sequenced. The plant expression vector is a modified pCambia vector (Guangzhou Jisai Biotechnology Co., Ltd.), the HPT II gene of the vector is cleaved with XhoI and replaced with the NPT II gene (the designed primers were amplified from the pBI121 vector, and the primers were designed at both ends with XhoI site), restriction enzyme digestion and sequencing results verified the direction of the 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 two elements. Finally, these two elements were respectively ...

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Abstract

The invention belongs to the field of plant genetic engineering, and in particular relates to protein CYP634A1 like-1 specifically expressed in fiber cells and application of the protein. The invention aims at solving a technical problem that the requirements on the fiber output and quality molecular design of improved cotton cannot be satisfied due to the lack of genes for improving specific or preferential expression of fiber at present. The amino acid sequence of the protein CYP634A1 like-1 disclosed by the invention is as shown in SEQ ID NO.3. Furthermore, the invention also provides a plant expression vector for expressing the protein. The invention offers a new choice for improving cotton fiber quality and cotton variety.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to protein CYP734A1like-1 specifically expressed in fiber cells and its application. Background technique [0002] Cotton is one of the world's major fiber crops. Cotton fiber cells are single cells formed by differentiated protrusions and polar elongation of epidermal cells in the outer integument of cotton ovules. Its growth and development process is divided into four distinct and overlapping periods: fibroblast initiation period, fibroblast elongation period, secondary wall thickening period and dehydration maturation period. Each period has its own characteristics, but adjacent developmental periods overlap. Each stage of fiber development has an effect on the yield and / or quality of fibers. The initiation of fiber differentiation determines the number of fibers in a single ovule. The rate and duration of fiber cell elongation mainly determine the length o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/84C12N1/21A01H5/00A01H6/60
CPCC12N9/0071C12N15/8205C12N15/8261
Inventor 罗明李芳隗廷翟云兰曾志锋裴炎肖月华侯磊李先碧
Owner SOUTHWEST UNIV
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