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Cotton pollen fertility related long-chain non-coding RNA and application of target gene of cotton pollen fertility related long-chain non-coding RNA

A long-chain non-coding and coding gene technology, applied in DNA/RNA fragments, applications, genetic engineering, etc.

Active Publication Date: 2021-06-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] TCONS_00473367 is specifically and highly expressed in flower buds of cotton fertile materials, and the target gene GhCYP724B encodes hydroxylase, which regulates the synthesis of brassinosteroids, but there is no report that this lncRNA and protein-coding genes regulate pollen fertility in cotton

Method used

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  • Cotton pollen fertility related long-chain non-coding RNA and application of target gene of cotton pollen fertility related long-chain non-coding RNA
  • Cotton pollen fertility related long-chain non-coding RNA and application of target gene of cotton pollen fertility related long-chain non-coding RNA
  • Cotton pollen fertility related long-chain non-coding RNA and application of target gene of cotton pollen fertility related long-chain non-coding RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, TCONS_00473367 and its target gene GhCYP724B expression analysis

[0076] Step 11 Acquisition and preservation of different cotton materials and different tissues

[0077] First, take the fertile material 2074B (Su Mian 20; Liu et al., Over-expression of transcription factor GhWRI1 in upland cotton. Biologia Plantarum 2018.62:335-342. The public can obtain it from China Agricultural University) at the seedling stage (Leaf), spore Flower bud from primary cell stage to pollen mother cell stage (FB-I), flower bud from meiosis stage to binucleate stage (FB-II), flower bud at pollen grain mature stage (FB-III), flower bud the day before flowering (Bud), full flower stage Petals (Petal), stigmas at full flowering stage (Stigma), anthers at full flowering stage (Anther), and seeds 10 days after flowering (Seed) were used for TCONS_00473367 tissue-specific expression analysis. In addition, flower buds from meiosis to binucleate stage of male sterile line 2074A, flow...

Embodiment 2

[0094] Example 2. Cloning of cotton long-chain non-coding RNATCONS_00473367 and protein-coding gene GhCYP724B fragment and construction of VIGS vector

[0095]Total RNA was extracted from flower buds of Gossypium hirsutum L. (Gossypium hirsutum L.) cultivar Su cotton 20 (2074B) from meiosis to binucleate stage, and cDNA was obtained by reverse transcription. R and VIGS724-F / VIGS724-R were used as primers for PCR amplification.

[0096] The primer sequences for the above-mentioned PCR amplification are as follows:

[0097] Primer VIGS67-F: 5'-CCTTAGGAGAAGAGGATGATTCGCT-3';

[0098] Primer VIGS67-R: 5'-TTCATCATGAAATCACAGTGAACCT-3'.

[0099] Primer VIGS724-F: 5'-TGGATTCTTTATTGGGTGGC-3';

[0100] Primer VIGS724-R: 5'-CCTGAAGTTTTGAACAAGGTGG-3'.

[0101] For the above PCR primer sequences, a Spe I (ACTAGT) restriction site sequence was added to the 5' end of the upstream primer, and a Pac I (TTAATTAA) restriction site sequence was added to the 5' end of the downstream primer.

...

Embodiment 3

[0108] Example 3, virus-mediated gene silencing cotton TCONS_00473367 and GhCYP724B

[0109] Step 31 Construction of recombinant Agrobacterium

[0110] The recombinant plant expression vectors pCLCrVA-TCONS_00473367 and pCLCrVA-GhCYP724B prepared in Example 2, as well as the positive control vector pCLCrVA-CHLI, the empty vector pCLCrVA and the auxiliary vector pCLCrVB were transformed into Agrobacterium tumefaciens GV3101 competent cells by the freeze-thaw method. The above-mentioned competent cells were purchased from Beijing Tuoyingfang Technology Co., Ltd., and after shaking culture in YEP medium without antibiotics for 3-4 hours at 28°C, they were coated on a medium containing 50 μg / ml kanamycin sulfate and 50 μg / ml rifampicin. Screening and culturing in YEP solid culture; through colony PCR, the PCR is carried out using primers VIGS67-F / VIGS67-R and VIGS724-F / VIGS724-R in Example 2 respectively, and then identifying positive single clones will identify the correct agricu...

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Abstract

The invention discloses a cotton pollen fertility related long-chain non-coding RNA and application of a target gene of the cotton pollen fertility related long-chain non-coding RNA. lncRNA and proteins provided by the invention are from upland cotton and consist of nucleotides as shown by a sequence 1 in a sequence table and amino acids as shown by a sequence 5 in the sequence table. Experiments prove that recombinant vectors pCLCrVA-TCONS00473367 and pCLCrVA-GhCYP724B containing DNA molecules shown from the 6th site to the 502th site in the sequence 1 of the sequence table and DNA molecules shown from the 884th site to the 1379th site in the sequence 3 of the sequence table are injected into cotton cotyledons, the vegetative growth of cotton plants is not affected, but the anthers wizen in the full-bloom stage, the number of pollen grains is reduced, and fertility is reduced. The invention provides a novel lncRNA / gene and method for creating a novel sterile line material, and has important significance in an aspect of creation of plant hybrids and utilization of heterosis.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the application of long-chain non-coding RNA TCONS_00473367 related to cotton pollen fertility and its target gene GhCYP724B. Background technique [0002] Cotton has obvious heterosis, and planting hybrid cotton can significantly increase cotton yield. Using the male sterility system to create hybrids saves time, labor and cost. Therefore, revealing the mechanism of male sterility in cotton is beneficial to the construction of male sterility system, hybrid breeding and heterosis utilization. [0003] Long non-coding RNA (LncRNA) is a kind of non-coding RNA with regulatory function, which has been proved to be involved in the regulation of male organ pollen fertility in a variety of crops. Rice LDMAR is an lncRNA with a length of 1236bp: under long-day conditions, sufficient abundance of LDMAR is a necessary condition for normal pollen development. The increased meth...

Claims

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Application Information

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IPC IPC(8): C12N15/113C07K14/415C12N15/29C12N15/82A01H5/00A01H6/60
CPCC12N15/113C07K14/415C12N15/8289
Inventor 华金平聂虎帅苏莹郭安慧程成
Owner CHINA AGRI UNIV
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