| China’s native tree species Larix olgensis has excellent traits such as early growth and strong adaptability,and its wide distribution has important economic and ecological value.At present,the tissue culture and genetic transformation system of Larix olgensis have been established,which is an ideal material for studying the molecular mechanism of conifers.WRKY transcription factors can play an important role in the response of plants to abiotic stresses such as drought,salt tolerance,high temperature and low temperature.This study cloned the LoWRKY1 gene of Larix olgensis,and carried out bioinformatics analysis on it;The expression of this gene under different abiotic stresses was analyzed by q RT-PCR technology,and the gene function was verified by transferring it to the heterologous plant tobacco and the native plant larch,and the physiological indexes under drought and salt stress were determined respectively.The results are as follows:(1)LoWRKY1 gene contains a CDS sequence of 1 035 bp,encoding 344 amino acids,and the encoded protein has a conservative WRKY domain domain,which is an unstable alkaline hydrophilic protein,containing 51 phosphorylation sites and 50 glycosylation sites.The results of evolutionary tree analysis show that they are closely related to North American spruce and ginkgo biloba.(2)qRT-PCR analysis showed that LoWRKY1 gene had the highest expression in the roots of Larix olgensis,the lowest in stems,and leaves.Under the Treatment of Na Cl,PEG and ABA,the expression of this gene in roots,stems and leaves varies,and preliminary analysis suggests that LoWRKY1 gene may be involved in the response when plants are subjected to abiotic stress,and the degree of response to different stresses varies in different tissue sites.(3)Construct recombinant vector VB191103-1905rcy-LoWRKY1,and transform it into Agrobacterium Gv3101 by thermal excitation method to genetically transform tobacco.The experimental results after Na Cl and PEG stress treatment showed that the heterologous expression of LoWRKY1 gene in tobacco enhanced the sensitivity of the photosynthetic system to adversity,the antioxidant enzyme(POD,SOD)activity was lower than that of the wild type,and the malondialdehyde(MDA)content was higher than that of the wild type,indicating that the salt tolerance and drought resistance of transgenic tobacco were worse than that of the wild type.(4)Agrobacterium conversion method was used to infect Larix olgensis callus,and the obtained transformants were GUS histochemically stained,and the colorless callus was stained blue,and the PCR at the DNA molecular level was positive.The results showed that the callus of the successful trans-LoWRKY1 was treated with Na Cl and PEG respectively,and the results showed that the activity of the wild-type callus protective enzyme SOD and POD was higher than that of the transgenic callus,and the MDA content was lower than that of the transgenic callus,and its growth state was better than that of the transgenic callus,and the results showed that the overexpression of LoWRKY1 reduced the stress resistance of embryonic callus.(5)The results of the body embryogenesis test of Larix olgensis embryogenic callus show that the amount of transgenic embryogenesis is higher than that of the wild type... |
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