Genome Analysis Of The Thermotolerant Transcription Factor Hsf Family Of Spinach And The Function Of SoHsfA1 Gene

Spinach（Spinacia oleracea L.） is an important green vegetable, which has high nutritional value and is widely cultivated in China.While the spinach has cold resistance but cannot tolerate high temperature, growing plants appear weak, low yields, poor quality, nitrate accumulation and other issues at temperatures above 25℃. The heat shock transcription factors（Hsfs） are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins（Hsps）, which are critical in the protection against stress damage. Therefore, studying of the Hsfs genes family is essential for understanding the spinach heat stress response mechanism. On the base of transcriptome and whole genome sequencing data of spinach, this paper carried out the features and function of SoHsfs, will provide a theoretical basis for the comprehensive analysis of the heat stress response mechanism of spinach and provide technical support for heat spinach molecular breeding.This paper completed the genome-wide identification of SoHsfs, cloned the full length CDS of all members. Based on the characteristics of their domain, SoHsfs were divided into A, B and C class, contructed phylogenetic tree of Spinach, Arabidopsis and Beta vulgaris which also belongs to Chenopodiaceae, analyzed and forecasted SoHsfs subfamily members and theirs functions, analyzed the heat stress expression pattern of the heat candidate core gene-SoHsfA1 using qRT-PCR. In order to further verify the function of SoHsfA1 in spinach, SoHsfA1 was transformed into E.coli（BL21） and Yeast（INVScl） to obtain recombinant BL21（pET28a-SoHsf A1） and recombinant INVScl（pYES2-SoHsfA1）. The study constructed spinach F₂ segregating population and identified its heat tolerance,which was preparation for the job of exploring the molecular marker linked to thermotolerance-related genes and the development of high-density molecular linkage map.Detailed results are as the following:1. 16 non-redundant Hsfs family gene members were identified in the genome of spinach. Analysis of SoHsfs domains showed that, DBD domains were highly conserved, NES domains were absent in some members, AHA domains existed only in part SoHsfA class members.2. The phylogenetic tree of Spinach and Arabidopsis Hsfs showed that the 16 SoHsfs were divided into four subgroups, including eight HsfAs, six HsfBs and one HsfCs.3. The phylogenetic tree of Spinach and Arabidopsis Hsfs has gaven each member of SoHsfs more detailed classification and function prediction so that we got two SoHsf A1 members, SoHsf5 and SoHsf16 which were similar to HsfA1 in Arabidopsis.4. Real-time Quantitative PCR results showed that, SoHsf5 and SoHsf16 genes were induced and expressed by the heat stress, and may play an important role in regulating the temperature response process.5. Constructing recombinant E.coli BL21（p ET28a-SoHsfA1） and Yeast INVScl（pYES2-SoHsf A1） for resistance analysis, and experiments showed either recombined SoHsf5 or SoHsf16 improved the tolerance of E.coli and Yeast under high temperature, NaCl, Osmotic stress. It is indicated that both SoHsf5 and SoHsf16 responsed the high temperature, NaCl, Osmotic stress, and the So Hsf16 was higher resistance than SoHsf5.6.The study constructed segregating F₂ spinach population including 215 individuals. The analysis of their heat tolerance showed that there was significant difference among heat tolerance of F₂ plants. F₂ individuals were divided into three groups named Thermostable, Intermediate and Thermolabile. Comparative analysis showed significant transgressive segregation of parental physiological index after heat treatment.