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Clematis florida thumb CLHSP18 gene coding sequence and application thereof

A clematis, sequence technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of yellowing leaves, susceptibility to blight, and shedding of stems

Pending Publication Date: 2019-08-16
SHANGHAI BOTANICAL GARDEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past 5 years (2014-2018) in Shanghai area, the continuous high temperature (above 35°C) in summer lasted for about 1 month, which seriously affected the growth of clematis, such as leaves turning yellow, wilting, falling off, and stems are easy to grow. Fusarium wilt, which limits the large-scale promotion and application of clematis in Shanghai

Method used

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  • Clematis florida thumb CLHSP18 gene coding sequence and application thereof
  • Clematis florida thumb CLHSP18 gene coding sequence and application thereof
  • Clematis florida thumb CLHSP18 gene coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning of CLHSP18 Gene from Clematis clematis

[0045] 1. The heat-resistant clematis cultivar 'Polish Spirit' (Shanghai Botanical Garden) was heat-shocked at 38°C for 3 hours, and the total RNA of young leaves was extracted. The extraction kit was the plant RNAout kit (Mianyang High-tech Zone, Sichuan Province). Tianze Genetic Engineering Co., Ltd.), total RNA was reverse-transcribed into cDNA using a reverse transcription kit (TaKaRa, Dalian, China).

[0046]2. Design degenerate primers according to the homologous sequence of rose RcHSP17.8 (accession number: EF053229.1) and dodder CjHSP 17.6 (accession number: AB017273), forward primer: 5'-CATTCRCTGAATHTCCTG-3' (SEQ ID NO.3 ) Reverse primer: 5'-AACMTCCTGGAGKACTTG-3' (SEQ ID NO.4).

[0047] A 247bp band was amplified from the 'Polish Spirit' cDNA by RT-PCR. The PCR product was recovered, connected to T-Vector (TaKaRa, Dalian, China), transformed into Escherichia coli DH5α, ((TaKaRa, D9057A)), and sequenced. The se...

Embodiment 2

[0075] Sequence Information and Homology Analysis of ClHSP18 Gene from Clematis clematis

[0076] The ClHSP18 gene obtained in Example 1 is 471 bp in length, and the detailed sequence is shown in SEQ ID NO.1. The polypeptide encoded by the gene consists of 156 amino acid residues, the molecular weight is 17959.49 Daltons, and the pI is 8.113. See SEQ ID NO.2 for the detailed sequence.

[0077] The coding region sequence of ClHSP18 full-length gene and its encoded protein sequence were nucleated in Non-redundant GeneBank+EMBL+DDBJ+PDB and Non-redundant GeneBank CDStranslations+PDB+SwissPort+Superdate+PIR databases with BLAST program Nucleic acid and protein homology detection, it was found that it has a high homology with rose (Rosa chinensis) RcHSP17.8. At the nucleotide level, it has a certain homology with the mRNA coding sequence (GeneBankAccessionNo.EF053229.1) of the Chinese rose (Rosa chinensis) RcHSP17.8 gene (see Table 1), and at the amino acid level, it has a certai...

Embodiment 3

[0079] Analysis of ClHSP18 Expression Pattern in Clematis clematis

[0080] Detected by RT-PCR

[0081] The total RNA was extracted from young leaves of 'Polish Spirit' (Polish Spirit) and 'StolwijkGold' (Shanghai Botanical Garden) at 25°C (normal temperature) and 38°C heat-shocked for 3 hours, and reverse transcribed into cDNA, and then A pair of primers were designed according to the full-length sequence of clematis ClHSP18 gene, forward primer 5'-ATGTCGCTTATCCTAAGT-3', reverse primer 5'-TTACGCAGTAAGATCAAT-3', and PCR was performed. After electrophoresis, analysis was performed with relative quantification software (Tiancheng Company). The results showed that the ClHSP18 gene was not expressed in the two clematis cultivars at room temperature, but expressed in 'Polish Spirit' but not in 'Kingstorvek' after heat shock ( figure 2 ).

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Abstract

The invention discloses a clematis florida thumb CLHSP18 gene coding sequence shown as the SEQ ID NO.1, an amino acid sequence shown as the SEQ ID NO.2, a recombinant expression vector containing a polynucleotides chain as shown in the SEQ ID NO.1 and host cells containing the recombinant expression vector. Heterogeneic expression of the clematis florida thumb CLHSP18 gene in arabidopsis thalianacan be realized, and the resistance to heat shock stress in the arabidopsis thaliana is improved.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to the coding sequence of clematis CLHSP18 gene and its application. Background technique [0002] Due to the increasingly obvious phenomenon of "greenhouse effect", the global temperature continues to rise, and the high temperature in summer has become the main environmental factor restricting the growth and development of plants. Plant growth and development are facing severe challenges of high temperature adversity. Heat shock proteins (heat shock proteins, HSPs) are a class of proteins that are expressed in large quantities after organisms are stimulated by high temperature and other stresses. According to their molecular weight, they are divided into HSP110s, HSP90s, HSP70s, HSP60s and small molecule heat shock proteins (smHSPs). Heat shock proteins are essential components for short-term adaptation of plants to adversity stress, and play a significant role in alleviating...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/82C12N1/21A01H5/00A01H6/20
CPCC07K14/415C12N15/1096C12N15/8271C12Q2531/113C12Q2549/119
Inventor 蒋昌华
Owner SHANGHAI BOTANICAL GARDEN