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Hordeum vulgare calmodulin gene HvCAM1 and salt tolerant application thereof

A calmodulin and gene technology, which is applied to the barley calmodulin gene HvCAM1 and its salt tolerance application field to achieve the effects of enhancing salt tolerance, mitigating adverse effects and improving stress resistance

Active Publication Date: 2018-02-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Barley has the strongest salt tolerance among cereal crops, but there is no report on barley CaM

Method used

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  • Hordeum vulgare calmodulin gene HvCAM1 and salt tolerant application thereof
  • Hordeum vulgare calmodulin gene HvCAM1 and salt tolerant application thereof
  • Hordeum vulgare calmodulin gene HvCAM1 and salt tolerant application thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 barley calmodulin HvCaM1 gene and promoter cloning

[0031] 1. Processing of plant material

[0032]Barley Golden Promise Seeds with 2% H 2 o 2 After sterilizing for 20 minutes, use ddH 2 After 5 times of washing, the seeds were germinated in the germination box with the ventral side up. Cultivate in the dark in the culture room (14h / 10h, 22°C / 18°C) for 3 days, until the aboveground part is 2cm white, then fill in the light. Seven-day-old barley seedlings were taken, frozen in liquid nitrogen and stored at -70°C for DNA and total RNA extraction.

[0033] 2. Extraction of barley genomic DNA

[0034] Barley genomic DNA was extracted by CTAB method.

[0035] 3. Extraction of barley total RNA

[0036] Total RNA was extracted by Trizol method. Genomic DNA was removed with DNase I enzyme (Takara, Japan).

[0037] 4. Synthesis of cDNA

[0038] Using Takara's The RT reagent Kit Perfect Real Time (DRR037A) kit was used to reverse transcribe the total RNA...

Embodiment 2

[0058] Example 2 RNAi and overexpression transgenic methods verify HvCaM1 gene function

[0059] 1. Construction of pBract214: HvCaM1 overexpression vector

[0060] Adopt the method of Gateway (Invitrogen, the U.S.), at first take the positive T vector containing cDNA in the above-mentioned embodiment 1 as template, design OE-HvCaM1-F / R primer amplification length is to cover the 450bp fragment of HvCaM1 coding region, primer information for:

[0061] OE-HvCaM1-F:5'-ATGGCGGACCAGCTCACC-3', (SEQ ID No: 11)

[0062] OE-HvCaM1-R:5'-TCACTTGGCCATCATGACC-3'. (SEQ ID No: 12)

[0063] After two rounds of PCR amplification, the BP Clonase TM II Enzyme Mix kit (Cat.No.11789-020, Invitrogen, U.S.) imports the fragment into pDONR TM In the / zeo vector (LifeTechnology), Escherichia coli DH5α was transformed and sent to the company for sequencing. Select the positive carrier containing the target fragment with correct sequencing, and then pass LR Clonase TM II Enzyme Mix Kit (Ca...

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Abstract

Belonging to the technical field of biological genetic engineering, the invention in particular relates to a hordeum vulgare calmodulin gene HvCAM1 and salt tolerant application thereof. The hordeum vulgare calmodulin gene HvCAM1 has a nucleotide sequence shown as SEQ ID No:1 and a full-length genome sequence 2124 bp. According to the invention, firstly a hordeum vulgare calmodulin coded gene is obtained by cloning and is named as HvCaM1, and the transcript cDNA full-length nucleotide sequence is shown as SEQ ID No:2, and the full-length cDNA sequence is 728 bp. The invention also utilizes RNAi interference method to silence the HvCAM1 gene successfully in hordeum vulgare Golden Promise, also utilizes the material to verify the negative regulation effect of HvCAM1 gene under salt stress can effectively enhance the salt tolerance of hordeum vulgare. At the same time, overexpression of the HvCAM1 gene in hordeum vulgare Golden Promise cannot enhance the salt tolerance of hordeum vulgare.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a barley calmodulin gene HvCAM1 and its salt-tolerant application. Background technique [0002] Salt stress is one of the most important abiotic stresses limiting crop growth and production worldwide. In the process of long-term adaptation to salt stress, plants sense external stimuli, generate and transduce corresponding adversity signals, thereby initiating various defense mechanisms to deal with damage caused by salt stress. Salt tolerance of plants is a complex quantitative trait. Although people have made great progress in the analysis of genes related to salt tolerance, the signal transduction and regulation mechanisms of related downstream genes are still unclear (Ligaba and Katsuhara, 2010, J Plant Res. 123:105-118). [0003] Ca 2+ It is generally considered as the second messenger in signal transduction in response to various environmental ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/113C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8273
Inventor 沈秋芳张国平吴德志叶玲珍傅良波
Owner ZHEJIANG UNIV
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