| Juglans regia is one of the famous four nut trees in the world with extremely high nutritional value. Walnut is widely planted in China and is one important economic nut trees at present. The research of the wanut tree were focuses on the walnut quality, breeding, processing and utilization of high-yield cultivation, rarely investigate the walnut physiological regulatory mechanisms in molecular level. In recent years, the global temperature variation causes the ‘cold spell’ in later spring happen frequently, which causes important impacts to the production and economic benefits of walnut. Small heat shock proteins(sHSPs) are important stress response proteins, however, there were few studies on them in woody trees, especial in walnut tree.In this study, several sHSP genes were screened from ‘Xiangling’ transcriptome; whose expression profiles exposed to various temperatures were analyzed by quantitative real time PCR(qRT-PCR). The infulence function of these genes on the yeast under cold, heat and salt was studied by contructing the yeast expression recombinant vectors, which was inserted into the yeast. Among these genes, JrsHSP17.3 was inserted into a plant expression vector and transient overexpression into the leaves of J. regia to varify the stress response role by analyzing the relevant physiological parameters under different conditions. The primarily results of this study were showed as follows:(1) Total fifteen sHSP genes were obtained by analysis of the walnut transcriptome. Sequences characteristic of these proteins were analyed by BLAST, evolutionary analysis and multiple sequence alignment. After comparion of the expression of these 15 sHSP genes by qRT-PCR, we found two of them showing much higher expression level, they were JrsHSP17.3 and JrsHSP20-1, and they were chosen for further analysis.(2) The expression profiles in tissues at different time points of JrHSP17.3 and JrHSP20-1 were analyzed by qRT-PCR, the results showed that these two genes can respond to high and low temperature stress, and the expression patterns of genes were different under different temperature or different stress time points with tissue and organ specific expression. For instcane, under low temperature stress, the expression of JrsHSP17.3 rendering upregulated with stress time expression in walnut roots; the amount of expression was 6.0fold of that under normal conditions at 6 h. And under salt stress, the expression was inhibited to 19% of the control in the root. The expression of JrsHSP20-1 in the root was 1.5, 4.0 and 42.6% of the control exposed to 6℃, 10 and 16℃ ℃, indicating that it has a certain tissue specific expression.(3) The JrsHSP17.3 and JrsHSP20-1 gene were indepandently insterted into a yeast expression vector pYES2 to constrcut recombinant vector pYES2-JrsHSP17.3 and pYES2-JrsHSP20-1, which were further transformed into the yeast strain INVSC1. The results showed that the yeast transformed by JrsHSP17.3 or JrsHSP20-1 improved the tolerance to abnormal temperature and salt stress, indicating that the two genes can be used as important candidate genes for improve plant resistance.(4) JrsHSP17.3 gene was inserted into the plant expression vector to construction prokII-JrsHSP17.3, which was used for transiently transformation into walnut leaves for stress response related analysis. The results showed that under low temperature(10℃), high temperature(40℃), salt(NaCl) stress treatment, the SOD and POD activities of the transient overexpression lines were significant higher that those of the control lines, while the reactive oxygen species(ROS)accumulation and MDA content of the transient overexpression lines were significant lower than those of the control lines, suggesting that the overexpression of JrsHSP17.3 gene obviously improved plant tolerance to salt and abnormal temperatures stresses. |
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