Molecular Cloning And Function Analysis Of Heat Shock Protein Genes SHSP7 And SHSP8 IN Gossypium Hirsutum L.

Biotic and abiotic stresses severely affect quality and quantity of crop.With the global warming,high temperature stress has become the first important problem.There are a number of low-molecular-weight heat shock proteins under high temperature stress,which are usually abundant and diverse in higher plants as oppose to other eukaryotes.The sHSPs range in size from 12 to 43 kDa and share a conserved C-terminal domain.In higher plants six nuclear gene families encoding sHSPs have been defined.Each gene family encodes proteins founding a distinct cellular compartment,including the cytocol,chloroplast,ER and mitochondrion.In general,the sHSPs are not found or few in normalvegetative tissues,but accumulate to high levels in response to heat stress.Recently,a salient process involved in the production of sHSP induced by heat shock and the relationship between sHSP and thermotolerance was proposed.There are some studies about tobacco,wheat,barely,soybean,corn,sorghum,red grape,tomato,carrot,potato,rice and mung bean in China and foreign countries,and these studies suggested that heat shock proteins(mostly the low-molecular-weight heat shock proteins)in plants improved the heat tolerance of plants and bacteria,but there are few reports about study of cotton.In our studies,we isolate the low-molecular-weight heat shock protein genes sHSP7 and sHSP8.Expression profile analysis and function analysis suggest that expression of the two genes are induced by high temperature and protect the proteins of plants.The main results are as follows:1.We design the degeneracy primers according homologous sequence and clone the middle fragment of the low-molecular-weight heat shock protein genes through RT-PCR,then 5' and 3' fragments are cloned by rapid amplification of cDNA ends(RACE).The full cDNA sequence is 723bp and ORF is 474bp which encodes a 17.9kDa protein with 157 amino acids.Further comparison shows its similarity is above 80%with the sHSP gene of Ricinus communis,Jatropha curcas,soybean and so on.We speculate that this gene is a low-molecular-weight heat shock protein,which is designated as sHSP7.Cluster analysis also suggests that sHSP7 has closed phylogenetic relationship with Ricinus communis,Jatropha curcas,soybean and so on.Structure analysis suggestes that sHSP7 has the highly conserved a Crystal domain.2.We design the degeneracy primers according homologous sequence and clone the middle fragment of another low-molecular-weight heat shock protein through RT-PCR,then 5' and 3' fragments are cloned by rapid amplification of cDNA ends(RACE).The full cDNA sequence is 750bp and ORF is 480bp which encodes a 18.6kDa protein with 159 amino acids.Further comparison showes it has high similarity with the sHSP gene of Ricinus communis,poplar,Jatropha and so on.We speculate that this gene is a low-molecular-weight heat shock protein,which is designated as sHSP8.Cluster analysis also suggests that sHSP8 has closed phylogenetic relationship with alfalfa,tomato,tobacco and so on.Structure analysis suggests that sHSP8 has the highly conserved ? Crystal domain.3.Expression analysis suggests sHSP7 and sHSP8 are induced by high temperature.They have the higher expression level in leaf than stem and root.The hours of heat treatment are 3,6,9,12,24,respectively.The result is that the expression level increases firstly,then decreases,finally increases again.Besides,the sHSP7 gene is induced by low temperature,but the expression level is rather low.The sHSP8 gene is not induced by low tepperature.4.We construct the over-expression vectors,transfer the two genes into tobacco,to study the function of the two genes.The result shows that transgenic tobaccos have higher tolerance than wild tobaccos under high temperature stress.In conclusion,the two low-molecular-weight heat shock protein genes are expressed under high or low temperature stress.The expression level under high temperature stress has a apparent increase,but the expression level under low temperature stress has no significant increase.Transgenic tobaccos suggest that the two genes play an important role in response to high temperature stress.The study supports the two genes will be widely used in cotton breeding under high temperature stress.