马铃薯甲虫热激蛋白基因Ld-hsp70的克隆及温度胁迫下的表达特性

doi:10.3864/j.issn.0578-1752.2021.06.008

Abstract

Abstract:

【Objective】 Heat shock protein (HSP), a class of highly conserved proteins, is generally found in all the organisms, which plays an important role in response to stress resistance. In order to ensure the normal physiological activities of the organism, the expression of HSP can be generated rapidly under the adverse environmental conditions. The objective of this study is to clone heat shock protein 70 (HSP70) genes of the important quarantine pest Leptinotarsa decemlineata (Ld-hsp70s), analyze their expression characteristics under temperature stress, and to clarify the function of L. decemlineata HSP70 under temperature stress. 【Method】The cDNA sequences of HSP70 genes of L. decemlineata were obtained based on the NCBI database. The full length cDNAs encoding HSP70 genes of L. decemlineata were cloned by RT-PCR and RACE technology. The full-length sequences were spliced by DNAMAN software. The sequence characteristics of Ld-hsp70s were analyzed by bioinformatic methods. The phylogenetic tree with the homologous sequences of HSP70 from L. decemlineata and other insects was constructed using the neighbor-joining (NJ) method with MEGAX software. The expression profiles of Ld-hsp70s were analyzed by qRT-PCR. The specific primers were designed using Primer 5.0 software. 【Result】The cDNA sequences of three HSP70 genes of L. decemlineata were obtained from the NCBI database, and were named as HSP70a, HSP70b and HSP70c, respectively. Three full-length sequences were obtained by cloning and splicing, and were named as Ld-hsp70a, Ld-hsp70b and Ld-hsp70c, respectively. The GenBank accession numbers were KC544268, KC544269 and KC544270, respectively. Sequence analysis showed that complete conserved domain, three signature sequences of HSP70 family, signal sequences and motifs of subcellular location at the carboxyl (C)-terminal were found in amino acid sequences of the three Ld-hsp70s. The conserved EEVD motif in the C-terminal of Ld-hsp70a and Ld-hsp70c indicated that they were cytosolic HSP70. The conserved KDEL motif in the C-terminal of Ld-hsp70b indicated that it was endoplasmic reticulum HSP70. Phylogenetic tree analysis showed Ld-hsp70a and Ld-hsp70b were clustered together with HSP70 from other insect species, while Ld-hsp70c was clustered together with the reported HSP70 from L. decemlineata. Ld-hsp70a, Ld-hsp70b and Ld-hsp70c had highest homology with Propylaea japonica Pj-hsp70, Lissorhoptrus oryzophilus Lo-hsp70, and Colaphellus bowringi Cb-hsp70, respectively. qRT-PCR results showed that the expression of all three Ld-hsp70s could be induced by high and low temperatures. No significant difference was observed in the relative expression of the three Ld-hsp70s after exposure to different temperatures for 1 h. The relative expression level of Ld-hsp70a was significantly upregulated by 2.24 and 2.41 folds after exposure to -10℃ and 44℃ for 4 h in female and male of L. decemlineata, respectively. However, no significant difference was observed in the relative expression of Ld-hsp70b and Ld-hsp70c under temperature treatments for 4 h. Whether Ld-hsp70s in female or male, no significant difference was observed of different treatment times at the same temperature. 【Conclusion】Ld-hsp70a in L. decemlineata can respond to temperature stress and may play an important role in the adaptation to adverse temperatures. Ld-hsp70b and Ld-hsp70c are not very sensitive to temperature stress, suggesting functional differentiation of the three Ld-hsp70s in response to abiotic stress.

Key words: Leptinotarsa decemlineata, temperature stress, heat shock protein 70, expression profile

HaiXia ZHENG,YuLin GAO,FangMei ZHANG,ChaoXia YANG,Jian JIANG,Xun ZHU,YunHui ZHANG,XiangRui LI. Cloning of Heat Shock Protein Gene Ld-hsp70 in Leptinotarsa decemlineata and Its Expression Characteristics under Temperature Stress[J].Scientia Agricultura Sinica, 2021, 54(6): 1163-1175.

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References 47

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引物名称 Primer name	引物序列 Primer sequence (5°-3°)	引物功能 Function of primers	退火温度 Annealing temperature (℃)
hsp70aF1	GCCATGAACCCCAATAACAC	hsp70a基因片段扩增 Amplification of hsp70a gene fragment	56.0
hsp70aR1	AGATGGTACCGGCATCTTTG		56.0
hsp70aF2	AGGAAGGACTTGACGAGCAA		56.0
hsp70aR2	TTTCTGCACCTTGGGGATAC		56.0
hsp70aF3	CAAGATCACCATCACCAACG		56.0
hsp70aR3	GATGGGGTTGCAGATGTTCT		56.0
hsp70aF4	CATACCTGGGCAAGACCG		56.0
hsp70aR4	CCAAAGACAGTGGCGTGA		56.0
hsp70bF1	GATGTTGGCACAGTCATTGG	hsp70b基因片段扩增 Amplification of hsp70b gene fragment	56.0
hsp70bR1	TGCTGAACAGTGGAGTCTGTG		56.0
hsp70bF2	AGCCAAGAGAGCCTTGTCTG		55.0
hsp70bR2	CAACGAGGACGATTTCATCA		55.0
hsp70cF1	GGCATTGATCTTGGAACCAC	hsp70c基因片段扩增 Amplification of hsp70c gene fragment	56.0
hsp70cR1	CTTCCTGGATCTTGGGATCA	hsp70c基因片段扩增 Amplification of hsp70c gene fragment	56.0
hsp70aF1	CAAGATCACCATCACCAACG	hsp70a 3° RACE扩增 3° RACE of hsp70a	56.0
hsp70bF1	TGATGAAATCGTCCTCGTT	hsp70b 3° RACE扩增 3° RACE of hsp70b	52.0
hsp70bF2	CAGCTCCCAGAGGTGTTC	hsp70b 3° RACE扩增 3° RACE of hsp70b	52.0
hsp70cF1	GCCAGGTAGCCATGAATC	hsp70c 3° RACE扩增 3° RACE of hsp70c	52.0
hsp70aFQ	ACGGTGACAAGTCTGAGGAGGT	实时荧光定量PCR qRT-PCR	60.0
hsp70aRQ	TGGTGGTGTTACGCTTGATGAG		60.0
hsp70bFQ	AGCAGCTATTGCCTATGGTTTG		60.0
hsp70bRQ	TTCCTGATGTCTTTGCCCTTC		60.0
hsp70cFQ	AACCAGTGGAGAAGGCTCTAACA		60.0
hsp70cRQ	TGTACTGCCGCACCATAAGC		60.0
GADPH-F	GATTCCACTCACGGACGATT	内参基因扩增 Amplification of the reference gene	60.0
GADPH-R	CATATTTGCCCCAAGGAATG		60.0
TUB-F	CTGCCTCTTTGAGGTTCGAC		60.0
TUB-R	TTACTGGGGCGTAAGTGACC		60.0

Cloning of Heat Shock Protein Gene Ld-hsp70 in Leptinotarsa decemlineata and Its Expression Characteristics under Temperature Stress

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