Cloning And Function Identification Of New Genes Related To Salt Tolerance From Chenopodium Album L.

Drought and soil salinization are the two main factors which affect agricultural production and this phenomenon is becoming increasingly severe. Researches have long been focused on improvement of the salt tolerance ability of the crops, and consequently to exploit and utilize saline-alkali soil properly by the way of genetic engineering. The screening and cloning of the function-determinate salt-related genes are the prerequisites.In the present study, based on the results of the subtractive suppression hybridization (SSH) library of Chenopodium album L. under salt stress from our previous results, 9 ESTs which were up-regulated under salt stress were selected by semi-quantitative RT-PCR from the novel and unknown EST function group. RACE (Rapid Amplification of cDNA Ends) method was employed to get the full length of cDNA sequences of those ESTs. Bioinformatics software was used to analyze and predict the functions of the obtained sequences. The full length cDNA sequences of ESTs were amplified by specific primers. Two ORFs of the putative genes from EST05 (named as P5P) and EST646 (named as TI) were cloned, and the plant expression and RNAi vectors were constructed, and Arabidopsis thaliana was transformed with these two genes to identify their contribution to plant salt tolerance. The results were as follows:(1) Both 5′and 3′end sequences of 6 ESTs (including EST05, EST14, EST179, EST340, EST544 and EST646), only 3′end sequences of 2 ESTs (including EST04 and EST140) and only 5′end sequence of EST702, were obtained by RACE method. Bioinformatic analysis revealed: the sequence from EST04 matched with chlorophyll a/b binding protein gene. EST05 had high similarity with predicted phosphoinositide 5-phosphatase phosphate gene of Ricinus communis. EST544 and EST646 shared the same sequence which had high similarity with the predicted trypsin inhibitor gene of Populus trichocarpa. EST140 and EST340 matched with function-unknown putative protein genes. There had no hit of EST14, EST179 and EST702 with known or unknown putative protein gene in blastn and blastx.(2) Full length (putative ORF) of EST05 and EST646 were amplified from cDNA of Chenopodium album L.. The sequencing results showed that the ORF of EST05 included 2042 nucleotides and encoded 607 putative amino acids; ORF of EST646 presented with two isoform (with different length), the longer ORF included 659 nucleotides and encoded 212 putative amino acids while the shorter ORF included 631 nucleotides and encoded 203 putative amino acids.(3) The plant over-expression and interference vectors of P5P (short for predicted gene from EST05) and TI (short for predicted genes from EST646) genes were constructed. T1 generations of transgenic Arabidopsis thaliana with these two genes were obtained.In the present study, the ORF of two putative genes related to salt tolerance were successfully obtained by RACE technique. The plant over-expression and interference vectors with these two genes harboring were constructed, and Arabidopsis thaliana was transformed. The T1 transgenic offsprings were obtained, and the identification of transgene is in progress. This work will lay a good foundation for later identification of gene function, and will provide some clues for salt-tolerant genetic engineering and improvement of soil salinization.