The Analysis Of Abiotic Stress Resistance Of Transgenic Populus Davidiana Dode×P.bollena Lauche Overexpressing ThLTP Gene

Lipid transfer protein is an important defense protein in plants, plays an important role in the resistance and adapt to stress. ThLTP from cDNA library was analyzed by bioinformatics, the gene has a total length of 635bp, the 5'non-coding region is 129 bp,3'non-coding region is 155 bp, the ORF (open reading frame) is 351 bp, encoding 116 amino acids. Its expected molecular weight is 22.76 kD, theoretical isoelectric point is 5.61.We studied the expression pattern by using real-time RT-PCR, and the results suggested that ThLTP was response to NaCl, ABA, PEG6000, and heavy metal septum (Cd) stress. it was up-regulated, and mRNA transcript level of this gene was higher in roots than leaves; while cold stress can suppressed the expression of ThLTP, the relative expression levels is lower than that of control, we speculated that ThLTP is a gene related to NaCl, PEG and CdCl2, induced by ABA.The ThLTP gene was inserted into the prokaryotic expressive vector pET32a and named BL21 (pET32a-LTP). And the highest expression system is 30℃,0.2 mmol/LIPTG induction 5 h. And then the recombinant BL21 (pET32a-LTP) were under stress. The results showed that NaCl and PEG stress on the growth curve analysis showed that the recombinant strain BL21 (pET32a-LTP) can significantly improve the Escherichia coli the ability to withstand abiotic stress, confirmed ThLTP was a drought and a salt tolerant genes.To further study the function of the ThLTP gene under salt stress, ThLTP gene were integrated into Populus davidiana Dode×P. bolleana Lauche via genome by Agrobacterium mediated method. It was totally obtained 20 independent lines with tolerance to kanamycin. Among the twenty, the expected 351 bp amplification product was detected in 10 lines. These nine transgenic lines (S7,S9,S12,S13,S14,S15,S19,S30,S44) were selected from the ten putative transformants for further analysis. The results of PCR and northern blot analysis indicated that ThLTP was expressed in the nine transgenic lines, and had integrated into the plant genome and could successfully transcript. The transgenic and thenon-transgenic plants were cultured on the medium with 0 mmol/L,50 mmol/L,100 mmol/L,150 mmol/L and 200 mmol/L NaCl for 20 days. Growth of transgenic plants was better than non-transgenic plants under Salt-stress conditions. The comprehensively analysis of leaf differentiation rate, average weight, rooting rate, salt injury index on the resistance to salinity indicated that the salinity resistance degree is S14> S19> S9> S30> S12> S7> S44> CK. Assay of salt stress of transgenic and non-transgenic plant showed that the chlorophyll contents of non-transgenic plants were significantly lower than those of transgenic plants under 0.8% NaCl after 3 d and 4 d, the average of chlorophyll contents was 79.3% higher than the control. The relative electrical conductivity of each transgenic line was significantly lower than that of non-transgenic plants, the average of chlorophyll contents was 28.2% lower than the non-transgenic plant. The experiments reflected that, under salinity stress, the transgenic plans suffered far less damage than control. Among them, S14 showed highest resistance and S9,S19 are inferior to it. The results also reflected that the over-expression of the gene in transgenic plants was beneficial for protecting membrane system.The expression of ThLTP gene strengthen the abilities of the salinity resistance.