Molecular Cloning And Expression Analysis Of Heat-shock-protein 70(HSP70) Genes From Gracilaria Lemaneiformis Under Heat Shock

Gracilaria lemaneiformis is an important economic red alga which can produce Agar. The original production area is Shandong Province and Liaoning Province, and now the areas are distributed across Guangdong, Fujian, Zhejiang and other places. G. lemaneiformis 981 is the main strain which is cultivated in the south coasts of China. Although the enduerable temperature is up to 26°C, yet the G. lemaneiformis 981 still can not successfully survive in the summer in the natural water of the South. So it is urgent to breed new strain which can tolerate much higher temperature. To breed the temperature- tolerance new strain, it is necessary to study the mechanism of G. lemaneiformis response to heat stress.Heat shock proteins (HSPs) are the protein family which can increase the tolerance ability when the body suffers the stress. They play a significant role in reducing the harm of stress on the body. HSP70 is an important component of HSPs and is one of the key factors to raise the thermostability of organisms. In our lab, EST of hsp70 was screened from the high temperature stress differentially expressed cDNA library, which indicated that hsp70 also played an important role in G. lemaneiformis to respond the heat stress. In this study, hsp70 was cloned from 981-type and wild-type by using RACE and Genome Walking method. This is the first time that the full-length sequence of hsp70 genes (hsp70-1 and hsp70-2) was cloned from G. lemaneiformis. The amino acid sequence was deduced from the cDNA sequence by the software DNAMAN, and the structural feature and phylogenetic position was analysed by bioinformatics. At last, mRNA expression level of hsp 70 of 981-type and wild-type G. lemaneiformis under different heat stress was studied by quantitative RT-PCR. The main results are as follows:Heat shock protein 70 genes (hsp70-1 and hsp70-2) were cloned from G. lemaneiformis. There are three typical HSP70 family signature sequences, TATA Box, CAAT Box and PloyA sequences in the obtained hsp70 genes. The full length DNA of hsp70-1 is 2582bp and having only one extron. The open reading frame of hsp70-1 is 1977bp, encoding 658 amino acids and the calculated molecular mass is 71.69kD. The length of 5' UTR is 484bp and 3' UTR is 121bp. The full length DNA of hsp70-2 is 2843bp and having only one extron too. The open reading frame of hsp70-2 is 2001bp, encoding 666 amino acids and the calculated molecular mass is 72.88kD. The length of 5'UTR is 730bp and 3' UTR is 112bp. There is an endoplasmic reticulum sequence- HDEL in hsp70-2. The similarity between hsp70-1 and hsp70-2 is 69% by the NCBI Blastn. Molecular phylogenetic analysis showed that the homology of HSP70-1 between Gracilaria lemaneiformis and Pophyra yezoensis is the highest (92%). And the highest homology of HSP70-2 between Gracilaria lemaneiformis and Monosiga brevicollis is 77%.After heat shock (28°C and 32°C) , the Realtime-PCR was performed to test the expression of hsp70 mRNA. The results showed that: in the heat-shock group of wild-type, the expression of HSP70-1 increased in the short time after heat shock. The expression of HSP70-2 was always lower than the control group. And the expression level of group at 32°C was lower than the group at 28°C. In the heat-shock group of 981-type, the expression of the two proteins were at the same trend of increase- decrease- increase. This suggests that the increase of HSP70 expression under heat stress is not sustained, but periodical. When the heat shock temperature is higher, the expression peak arrives earlier and greater, and the expression level will be higher with the heat shock time prolonged. In addition, the expression level of HSP70-1 is higher than HSP70-2 under heat stress, which suggested that HSP70-1 played a leading role and HSP70-2 played a supporting function. With comprehensive comparison of the HSP70 expression of two kinds of G.1emaneiformis under heat stress, the expression level of HSP70 of G. 1emaneiformis 981 was obviously higher than that of wild-type and the expression time endured longer, which may be directly related to the higher temperature tolerance ability of G. 1emaneiformis 981. This study lays a molecular basis for revealing the mechanism of G. lemaneiformis responding to heat stress. And the study also provides the genetic information for breeding the heat -tolerance cultivar.