Cloning and Function Analysis of P-ATPases Genes in Cotton and Tomato
|School||Nanjing Agricultural College|
|Course||Crop Genetics and Breeding|
|Keywords||P-type ATPases clone Agrobacterium-mediated transformation promoter|
P-type ATPases are one kind of very important enzymes in Eukaryotes and many Prokaryotes and they are related to the growth, development and abiotic stress tolerance of plant. At present, there is no report about P-type ATPases gene in cotton and tomato. In this study, the P-type ATPase genes of tomato and cotton were cloned, and their expression was analyzed in different tissues and under different abiotic stress conditions. Moreover, the role of P-type ATPases was preliminarily studied in growth, development and stress tolerance of plants. It is believed that the results will be helpful to improvement of stress tolerance of cotton and tomato by genetic manipulation. The main results are as follows:1. Two ESTs of cotton homologic to P-type ATPases gene from Verticillium dahliae Kleb. was obtained through Blast in Gene Bank database. The full open reading frame of the gene was obtained by RACE and Genome walking. As a result, the entire coding region of P-type ATPases is 3570bp and encodes a polypeptide with 1190 amino acids. It shares about 90% homology to its homolog in Populus trichocarpa and Ricinus communis and named Gbpatp.Then, the translated product of Gbpatp was subcellularly localized in the cell membrane. The expression of Gbpatp in different organs of cotton plants was analyzed by semi-quantitative RT-PCR. The results indicated that Gbpatp was abundant in stem and fiber extremely, and it was in low level in seed, but scarce in leaves and flower bud. It showed Gbpatp might be closely related to the growth and development of stem, fiber and seed. The expression increased remarkably after treatment with PEG. After treatment with CuSO4, the expression increased as well, but it decreased in 48h compared with 24h. The expression increased after treatment with low temperature. In response to GA and ABA treatments, the expression stayed at a certain level in 24h and 48h. It is suggested that Gbpatp was related to stress tolerance of plants.To observe what will happen when the Gbpatp is over-expressed the expression vector of PCXSN-Gbpatp were constructed and used to transform tobacco. Twelve transgenic tobacco plants have been obtained.2. Two ESTs of tomato homologic to P-type ATPases gene from Verticillium dahliae Kleb. were obtained by searching in Gene Bank database. Then the full open reading frame of the gene was obtained by bioinformatics analysis and named Lepatp.The RNAi vector 2301-P1-P2 was constructed on the basis of Lepatp sequences and used to transform tomato. Eleven transgenic plants have been obtained up to now. There were 5 transgenic plants in which Lepatp was not detected by semi-quantitative RT-PCR. It implied that Lepatp was silenced in these plants. Further observation showed the silencing of Lepatp resulted in inhibition of the root growth. It indicated Lepatp was related to the growth of root.3. On the basis of the known sequences of tomato P-type ATPases, the promoter of Lepatp was cloned by the method of silico cloning. Then a recombinant plasmid with GUS gene was constructed and used to transform Arabidopsis thaliana. Four transgenic plants were obtained. The histochemical analysis showed that the GUS gene was expressed in vascular bundle of petiole, root tip, the bottom of flower and pod, but not in leaf. The results indicated it was a tissue-specific promoter.