Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (8): 1486-1494.doi: 10.3864/j.issn.0578-1752.2017.08.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Function Analysis of Heat-Shock-Protein LsHsp70-2711 Gene Under High Temperature Stress in Leaf Lettuce (Lactuca sativa L.)

LI YaBo, LI Ting, HAN YingYan, FAN ShuangXi   

  1. College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206
  • Received:2016-09-13 Online:2017-04-16 Published:2017-04-16

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Abstract: 【Objective】Through cloning of genes related to Hsp70, and using VIGS to analyze Hsp70 expression and morphological change of leaf lettuce under heat stress to lay a foundation for analysis of the response mechanism and molecular mechanism of heat shock protein under heat stress.【Method】The full-length cDNA gene of LsHsp70-2711 gene was obtained by homologous cloning and RACE technology. By real-time fluorescence quantitative PCR (qRT-PCR), the expression of the gene from P-S11and G-S59 was analyzed. According to the VIGS technology, the pTRV-LsHsp702711 vector was constructed and transformed into Agrobacterium GV1301. The leaves of lettuce were infected by the injection method. After three weeks, the positive plants were identified by PCR. The gene expression and morphology were compared between the control group and positive group, and the expression characteristics of LsHsp70-2711 were analyzed again after heat stress and drought treatment. 【Result】The sequence analysis indicated that the full-length cDNA was 2226 bp, the open reading frame was 2154 BP, encoding 718 amino acids, and its homology compared with that of Arabidopsis (NP_187864.1), Tomato (NP_001266213.1), Saussurea Medusa (AAB99745.1), and other species. qRT-PCR results showed that the expression of the gene in the two cultivars was up-regulated, and the expression level of heat resistant cultivars was significantly higher than the heat sensitive cultivar, and the gene expression of P-S11 was inhibited by the increase of stress time, and the expression level of G-S59 was higher in the heat resistant variety. In heat resistant variety, the maximum expression of LsHsp70-2711 appeared at 42℃, 60 min, and the maximum expression of heat sensitive variety reached at 37℃, 60 min. Prediction of subcellular localization showed that LsHsp70-2711 was mainly in the cytoplasm. The constructed vector was used to invade the leaves of lettuce, and the positive plants were obtained after identification. LsHsp70-2711 expression decreased after it was silenced by VIGS, compared with the control, the stem length of the positive plants was significantly increased. After heat stress and drought treatment, the expression of LsHsp70-2711 was significantly lower than the control, and the influence of high temperature treatment on LsHsp70-2711 was greater than drought stress.【Conclusion】 LsHsp70-2711 gene belongs to Hsp70 gene family, and is related to the heat resistance in lettuce. The research results of the study will provide theoretical supports for the analysis of function of high temperature in bolting of lettuce.

Key words: Lactuca sativa L., Hsp70, clone, temperature stress, VIGS

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