Expression Analysis And Functional Study Of Heat Stress Related Genes In Pepper

Pepper(Capsicum annuum L.) as an important vegetable crop, is deeply loved by a large population due to its major ingredient in cuisines, essential vitamins and other healthy nutrients. However, pepper is sensitive to high temperature, especially in reproductive stage. Once suffering continuous HS, the pollination and fertilization of pepper will be severely damaged, which can greatly reduce the yield and quality of pepper fruits. Therefore, illuminating the mechanism of pepper acquired thermotolerance will be helpful for hunting for the methods of improving the pepper thermotolerance, effectively reducing the heat stress' s damage to pepper. To achieve this goal, identification, analysis and studying the function of the heat stress related genes in pepper are the first step. In this research, we identified three heat stress related gene families, including heat shock transcription factor(Hsf), heat shock protein 70(Hsp70) and heat shock protein 20(Hsp20) family, analyzed the tissue-specific expression patterns of the gene family members, the response to abiotic stresses, studied the function of three genes from above three families, respectively.The major results are described as followings.1. There are 25 members in pepper Hsf(CaHsf) gene family, which can be divided into three classes based on the oligomerisation domains, class A, B and C, the number of class A members is largest, following by class B, and the number of class C is the smallest, including one member. The members of this gene family are distributed on11 out of pepper 12 chromosomes, no members are distributed on chromosome 11;the members share a highly conserved structure; the expression patterns of these genes are tissue-specific, and they express in at least one of five tissues, root, stem,leaf, pericarp and placenta; the members of CaHsf family respond to heat, salt andosmotic stresses, and also respond to exogenous calcium ion, putrescine, abscisic acid and methyl jasmonate.2. There are 35 members in pepper Hsp20(CaHsp20) gene family, their structures are conservative, and they are belong to 12 subfamilies; the members are distributed on pepper 12 chromosomes, the largest number of members are found on chromosome 3, 6 and 8, five members on each of the three chromosomes, and the chromosome 2, 11 and 12 have the least number of members, only one member on each of the chromosomes. Almost all genes have 0 or 1 intron, except for CaHsp27.6,which has five introns; the expression levels of the family genes in different tissues are low under normal condition, but are strongly induced by heat stress; under heat stress treatment, the expression peak of CaHsp20 s in thermotolerance line R9 is earlier than that in thermosensitive line B6; there are various stress response cis-elements in gene promoters, including heat shock elements.3. There are 21 members in pepper CaHsp70 gene family, and the structures of them are the most conserved; based on the cell localization, the CaHsp70 genes can be divided into four subfamilies, cytoplasm, endoplasmic reticulum, mitochondria and plastid subfamily, in which the number of cytoplasmic CaHsp70 members is the largest; the family members are distributed on 12 chromosomes; the number of gene introns is diverse, there are no introns in CaHsp70-2, CaHsp70-13 and CaHsp70-20,the number of the introns in CaHsp70-10(10) is the most, the number of introns in cytoplasmic subfamily members is 0 or 1; the promoters contain a variety of stress-related cis-elements, including heat shock elements, low temperature response elements, defense and stress response elements, and plant hormone response elements,the number of heat shock elements in CaHsp70-10 promoter is maximum; the gene expression of the family genes has tissue specificity, and is regulated by heat stress and Ca2+.4. Pepper CaHsfA2 gene is located in the nucleus and has transcriptional activity;there is no difference between the phenotypes of transgenic and wild type plants,CaHsfA2 is able to enhance the thermotolerance and salt tolerance of transgenic plants,and regulate the expression of many stress-related genes in transgenic lines. Itspromoter has basic activity, and is also induced by heat stress, low temperature and salt stress. Although lacking the activation domain and nuclear export signal, the N-terminal region(CaHsfA2?N) can enhance the thermotolerance of transgenicplants, but its function is weaker than that of CaHsfA2.5. Pepper CaHsp70-2 locates in the cytoplasm and nucleus. Overexpression of CaHsp70-2 does not affect the normal growth of transgenic plants. CaHsp70-2 can enhance the thermotolerance of transgenic plants, including the basal thermotolerance,acquired thermotolerance and the tolerance to moderately high temperature;CaHsp70-2 can reduce salt and drought tolerance. CaHsp70-2 is able to regulate the expression of stress-related endogenous genes in transgenic plants, including heat shock proteins, heat shock transcription factors, ascorbate peroxidase genes and galactinol synthase genes.6. Pepper CaHsp24.2 locates in mitochondria and cytoplasm, and may be also located in chloroplast. The growth of transgenic plants is not affected by overexpression of CaHsp24.2 gene. CaHsp24.2 can enhance the thermotolerance of transgenic plants, and regulate the expression of heat stress-related genes in transgenic plants. The expression levels of AtHsp17.6B-CI, AtHsa32, AtAPX2 and AtHsp90 are obviously regulated by overexpressing CaHsp24.2 gene.7. The pepper plants silenced CaHsfA2, CaHsp70-2 and CaHsp24.2 show little plant type and leaf malformation. After heat stress treatment, the thermotolerance of silenced plants is reduced compared with the negative control plants. There is an interaction relationship among the three genes, compared with CaHsp24.2 gene,CaHsp70-2 has a greater effect on the regulation of CaHsfA2; CaHsp70-2 is less affected by CaHsfA2, but is greatly affected by CaHsp24.2; the interaction between CaHsp24.2 and CaHsp70-2 is more stronger than that of between CaHsp24.2 and CaHsfA2.