Analyses And Functional Study Of SHSP Gene Family In Mulberry

Plants in nature affected inevitably by a variety of environmental stresses. Response to stresses, biological processes of plants including structural, physiological, biochemical and molecular changes occur rapidly. In the long-term evolution, plants develop and form a complex mechanism to adapt to a variety of environments.Plants produce a family of proteins known as heat shock proteins (HSPs) quickly under high temperature. HSPs act as molecular chaperons that can assit fold, assemble, locate and transport of nascent peptide. Small heat shock protein (sHSPs) is a kind of least conserved HSPs. sHSPs is most abundant in plants, have many varieties, complex structure and functional diversity. sHSPs associate with the unnatural proteins and prevent their irreversible aggregation, play an important role in plant resistance to heat stress.Mulberry is a kind of perennial woody plants. Mulberry leaves have long been culticated for silkworm rearing. In addition, mulberry has medicinal, edible and ecological functions. As an excellent ecological economic tree species, it is resistant to heat, drought, salt, and cold in some extent. With the completion of genome sequencing of mulberry, resistance research of mulberry has been developed from germplasm resources screening and physiology and biochemistry studies to the molecular level. In the present study, we identified 29 sHSP genes from the Morns genome database by bioinformatics analyses. The phylogenetic relationships, gene structure, transcriptional regulatory elements, and spatial expression pattern of mulberry sHSP genes were also investigated. The qRT-PCR was conducted to examine the transcriptional expression of 13 sHSPs under heat, cold, drought, and salt treatments in mulberry seedling (Morus indica cv. K2). Gene Mn16.8-C Ⅰ was chosen to study its epidermal subcellular localization in onion. The function and promoter activity of Mn16.8-C Ⅰ were further sudied. The main results of this study are as follows:1. Bioinformatics analyses of sHSP of mulberryWe identified 29 sHSP genes from the Morus genome database by informatics analysis. The length of amino acids were between 136～240 aa. The molecular weight of these genes were between 15.68～26.84 kD and PIs ranged from 4.49～9.34. The mulberry sHSPs were divided into 13 subfamilies and 1 orphan gene by analyses of phylogenetic relationships and subcellular localization. Of the 13 subfamilies, CⅫ and CⅩⅢ were two new subfamilies. All of 29 sHSP genes contain an ACD domain. Except for genes in CⅣ and CⅤ subfamily, remembers in other subfamily have β2-β9 folding chain. Four sHSP genes had a perfect HSE element. The expression of 29 sHSP genes was detected in root, bark, leaf, male flower, and winter bud with different levels.2. Cloning of sHSP genes expression analyses under abiotic stressMulberry (Morus indica cv. K2) was used to extract RNAs and cDNA was synthesized as templates. We then cloned 13 sHSP genes in this mulberry species. After sequencing, the primers for qRT-PCR were designed. The qRT-PCR was conducted to examine the transcriptional expression under high temperature, cold, drought, and salt of these genes. The results showed that 10 sHSP genes response to heat stress quickly. There were 5,9, and 7 sHSP genes respectively response to cold, salt, and drought.3. Subcellular localization and promoter activity of Mn16.8-C ⅠGene Mn16.8-C Ⅰ responsed to high temperature, cold, drought, and salt, suggesting it played an important role in plant resistances. Therefore,35S::Mn16.8-CI-EGFP and 35S::EGFP transient expression vector were constructed and transformed into onion epidermal cell by agrobacterium infection.The results indicated that Mn16.8-C Ⅰ localized in the nucleus and the cytosol. To study the activity of Mn16.8-C Ⅰ promoter, pBI12l-PMn16.8-C Ⅰ was constructed and transformed tobacco. The results showed that Mn16.8-C Ⅰ promoter express omission slightly under 25 ℃ and speculated that can drive the expression of GUS gene efficiently under 38 ℃.4. Function study of Mn16.8-C ⅠTo study the function of Mn16.8-C Ⅰ, p1GNL-Mn16.8-C Ⅰ was constructed and transformed tobacco. We measured the physiological indictors of tobacco under high temperature. The results showed that MDA content of transgenic tobaecco were lower than that of wild type under high temperature stress. The activity of antioxidant enzymes SOD and POD of transgenic tobacco were higher than those in wild type. That results indicated that overexpression of Mn16.8-C Ⅰ can improve the heat resistance of transgenic tobacco.