A New Material Cultivation Of NtLTP4 Transgenic Potato For Resisting Salt And Drought And Disease

The commercial value of potato(Solanum tuberosum L.)takes the forth place in all the plant global wide,just behinding wheat,rice and corn.The Agriculture Goverment has putted the project of potatoes being the staple food on the agenda,since 2015.Although the planting areas of potato are widely in China,the quality and average yield of potato are difficult to be improved duo to the environmental stresses.The environmental stresses can led potato to varying degrees of yield reduction,even up to 70%.It's reported that NtLTP4 encodes a non-specific lipid transfer protein,overexpressing NtLTP4 in tobacoo can improve the tolerance for biotic and abiotic stresses.Here,we overexpressed NtLTP4 in tomato and then got transgenetic lines with new phenotypes,such as salt-alkalescence tolerant,drought tolerant and resistance to bacterial wilt.The results and conclusions are as follows:(1)The expression activity analysis of ubi3 promoterTo generate the expression vector ubi3::pX6-GFP,a 921-bp DNA fragment from the region upstream from the ubi3 coding sequence was cloned into the pX6 vector.And then a DNA fragment encoding GFP was inserted into the vector.The expression vector ubi3::pX6-GFP was introduced into the leaves of Nicotiana benthamiana transiently by Agrobacterium-mediated transformation through injectors.Two days after injected,the fluorescence was observed,which indicates the ubi3 promoter can activate the expression of downstream genes.(2)Construcing of ubi3::pX6-NtLTP4 vector and obtaining of transgenic plantsWe putted the NtLTP4 into the vector ubi3::pX6 by T4 ligation and then got the expression vector ubi3::pX6-NtLTP4.The vector was introduced into potato by Agrobacterium-mediated transformation using floral-dip method and a total of 18 transgenic lines were obtained.Among these,15 transgenic lines were positive detected by RT-PCR.And then we detectedthe NtLTP4 expression level in these 15 lines by qRT-PCR.Results showed that the expression levels of L2,L9,L11,L12 and L13 were higher than others.Then Southern blot analysis was performed on the 5 lines,from the results,we can find that signals appear in all these 5 lines and there are 1 to 4 bands in these signals,indicating that NtLTP4 was successfully inserted into the genome,at least 1 to 4 copys.The result of Western blot about these 5 lines showed that the molecular weight of the protein is about 12 kDa were detected.All of these results suggested that NtLTP4 were expressed successfully in these transgenic lines.(3)Abiotic and biotic stress resistance identification of transgenic linesAccording to the above results,we chosed 3 transgenic lines(L11,L12 and L13)with higher NtLTP4 expression levels to conduct the experiments of salt stress,drought stress and bacterial infection treatment to detect their phenotypes.The results showed that the transgenic lines grew better than wild types in the high-salt environment,and the higher the expression of NtLTP4,the stronger salt tolerance of potatoes.The survival rate of transgenic lines under drought conditions were higher than the wild type,and the higher the expression level of NtLTP4,the higher the survival rate of potatoes and the better in drought tolerance.The incidence and degree of transgenic lines inoculated with bacterial isolates were lower than those of wild-type strains,and the higher the expression level of NtLTP4,the lower the incidence and degree of potato bacterial wilt,and the stronger the resistance to bacterial isolates.These results showed that overexpression of NtLTP4 could enhance the resistance of potato to abiotic stress(high salt,drought)and biotic stress(Ralstonia solanacearum),and the higher expression of this gene,the better tolerance of the plant to abiotic and biotic stresses.