Cloning And Function Research Of A DEAD-box Helicases Gene,SlDEAH1in Solanum Lycopersicum

The helicases are considered to be enzymes that present widely in all organisms.They utilize the energy of hydrolyzing a NTP （mainly is ATP） and catalyze theunwinding of double-stranded DNA （DNA helicases） or double-stranded RNA （RNAhelicases）. These kinds of proteinase exist widely in all most prokaryote and eucaryotesuch as virus, human being and so on. The research shows that DEAD-box helicasesplay a significant role in basic cell processes,including RNA metabolism, the regulationof gene expression, nucleocytoplasmic transport, the response of hormone signal,ribosome biogenesis and assembly and stress responses and so on. In addition,theDEAD-box helicases also can play important roles in the RNA folding and theprocessing of RNA-protein complex as the molecular chaperone.Tomato is used asmodel plant to study fruit ripening. Meanwhile, it is convenient to study the function ofgenes in toamto owing to the shorter growth period and the consummation of completegenome sequencing. A DEAD-box homologous protein was selected from the NCBIDatabase by means of program of homologous alignment according to the publishedDEAD-box protein in Arabidopsis thaliana and Maize and named SlDEAH1.Bioinformatics analysis indicated that it was the member of DEAH family ofDEAD-box helicases family.But there is no report about DEAD-box helicases intomato.And the concrete functions of tomato SlDEAH1gene are unclear.In order tostudying the funcition of SlDEAH1gene in growth and development, stress resistance,regulation of gene expression and fruit ripening and so on, we carry out the followingresearch:1. A DEAD-box homologous protein was selected from the NCBI Database byhomologous alignment according to the published DEAD-box protein in Arabidopsisthaliana and Maize and named SlDEAH1（accession number: XP₀04248028）.Thespecific primers which were used to clone the full-length cDNA of SlDEAH1gene weredesigned according to the sequence （accession number: XM₀04247980） of the selectedSlDEAH1gene. We cloned the full length of SlDEAH1gene using the cDNA of Bphases of the fruit of wild type （WT） tomato. The result of agarose gel electrophoresisindicated that a2300bp objective band around was cloned which in accord with ourprediction. This genetic fragment was turned out to be the target fragment after PCRproduct recovered and purified, cloning and sequencing. 2. The bioinformatics analysis were done according to the obtained SlDEAH1sequence. Sequence analysis indicated that the full length cDNA of SlDEAH1was2335bp, which open reading frame was2073bp, encoding690amino acid residues. Thesecondary structure prediction showed that the SlDEAH1protein had42.46％α-helix,6.81％β-strands and34.35％random coil,16.38%extended-chain. The subcellularstructure location indicated that SlDEAH1might locate at plasmalemma. The SlDEAH1protein had9conserved motifs, involving in ATPase, helicases and RNA Binding,respectively by homologous alignment. Conserved domains-search results displayedthat the SlDEAH1protein was the member of DEAD-box helicases family.3. The RNAi expression vector pBIN19-iSlDEAH1was completed by usingdouble digestion that utilized the intermediate vector pHANIBAL.4. The RNAi expression vector pBIN19-iSlDEAH1was transfered into the wildtype （AC++） tomato cotyledon explant via Agrobacterium-mediated transformation. Thepositive transgenic tomato lines were obtained by resistance screening, the detection ofNPT II, and the quantitive RT-PCR which was performed to determine the percentage ofthe ablation of SlDEAH1expression.5. The tissue-specific expression pattern of SlDEAH1in different tissues of WTtomato AC++,the Nr and rin mutants was studied using the Real-time PCR （RT-PCR）method which used the tomato housekeeping gene CAC as an inner reference. Theresults showed that the expression level of SlDEAH1was higher in the sepal, the matureleaves and the senile leaves of WT tomato and the highest in the fruit of B+4and B+7phases, and there is an obvious increasing trend of the SlDEAH1in the fruit ripeningand development. The expression level of SlDEAH1was decreased in turn in from the Bphases of the Nr and rin mutants.6. We made use of the Real-time PCR （RT-PCR） method to study the expression ofSlDEAH1under stress such as high temperature（40℃）, low temperature（4℃）,dehydration, injury and salt.The results indicated that the expression of SlDEAH1wasinduced at different degree by high temperature（40℃）, low temperature（4℃）,dehydration, injury and salt stress,but inhibited in root by salt stress.7. In order to study the influence of hormone on the expression of SlDEAH1, weused the IAA, ZT, GA3, ACC, MeJA,ABA to treat the Wild Type （WT） tomato AC++seedling. The results demonstrated that the expression of SlDEAH1was induced indifferent lever by ABA, ACC, IAA, GA3,meJAand ZT, but the most obvious inductionto its’ expression lever was ABA. 8. The results of henotypic analysis and statistical analysis indicated that theSlDEAH1gene playes important roles in the development of toamto leaves and mayplayes a certain role in the development of fruit. However, the gene has no effect onfruit ripening.The results suggest that SlDEAH1encodes a functional DEAD-box RNA helicaseand plays important roles in responses to Hormone treatment and in defence responsesagainst biotic and abiotic stresses, and also in the growing development of tomatoleaves. Furthermore, it may play a certain role in the development of tomato fruits.