Creation Of STTM394 Lettuce And Analysis Of Its Heat Resistance

Global warming has caused high temperature in many places,which has seriously affected crop growth.Lettuce is a kind of leafy vegetable that likes cold but not heat resistance.It is widely planted all over the world.However,due to its heat-resistant characteristics,the yield is seriously affected by high temperature.Therefore,improving the high-temperature resistance of lettuce has practical application value.miRNA is a kind of small noncoding RNA composed of 20-24 nt.It targets messenger RNA（m RNA）at the post transcriptional level and plays an important role in plant individual growth and development and resistance to abiotic stress.miR394 participates in the regulation of abiotic stress of plants by regulating the target F-box protein.It has been studied in Arabidopsis,tobacco,corn and other plants,but there is no research on miR394 response to high temperature stress in lettuce.Based on the efficient lettuce tissue culture system optimized in the early stage of the experiment,the constructed STTM-miR394 vector was transferred into（Lactuca sativa L）.through agrobacterium mediated technology,and the transgenic lettuce plant（STTM394Las）with down-regulated miR394 expression was obtained.With T₃ generation STTM394Las and wild-type lettuce（WT）as experimental materials,the morphology,physiology and to explore the effect and mechanism of reducing the expression of miR394 on high temperature resistance of lettuce.The main results are as follows:1.In this study,the gene sequence of lettuce miR394 was obtained through pre laboratory sequencing.STTM-miR394 vector was constructed by STTM technology.Transgenic lettuce plants with reduced miR394 expression were obtained through agrobacterium mediated and tissue culture.The expression of miR394 in T₃ transgenic lettuce decreased by 3.85 times and the expression of F-box increased by 2.07 times.2.STTM394Las has stronger morphological stability and photosynthetic ability under high temperature treatment.The morphological and photosynthetic indexes of STTM394Las and WT were measured;The results showed that the index changed more obviously under H2treatment.Compared with WT,STTM394Las increased plant height by 20%,root length by11.8%,leaf area by 14.8%,and higher biomass accumulation;Pn,Tr and Gs of leaves increased by 66.8%,3%and 8.6%respectively,the contents of chlorophyll a and chlorophyll b increased by 27.5%and 7.3%,and Ci decreased by 6.8%.3.Under high temperature treatment,STTM394Las has stronger antioxidant capacity and osmotic regulation capacity.In order to study the reason why STTM394Las maintained stronger morphological stability and photosynthetic capacity under high temperature treatment,the relevant indexes of antioxidant system and osmotic regulation system were measured in this experiment;The results showed that H2 treatment had more significant index changes.Compared with WT,the concentrations of H₂O₂ and MDA in STTM394Las leaves decreased by 45.8%and 29.1%respectively,and the activities of SOD,POD and CAT increased by 8.4%,13.8%and 15.3%respectively;The contents of soluble sugar and soluble protein increased by22.3%and 14.7%,and the relative water content of leaves increased by 6.8%.4.In order to explore the possible molecular mechanism of reducing miR394 and improving high temperature resistance of lettuce,q RT-PCR was used to detect the expression of miR394,target F-box and heat shock protein in lettuce under high temperature treatment;The results were as follows:under high temperature treatment,the expression of miR394decreased and the expression of target F-box,heat shock protein gene HSP60,HSP70 and HSP90 increased;Compared with WT,STTM394Las had lower expression of miR394 and higher expression of F-box,HSP70 and HSP90.The results showed that miR394 was negatively correlated with the expression of HSP70 and HSP90,and F-box was positively correlated with the expression of HSP70 and HSP90.In conclusion,STTM-miR394 vector was successfully constructed by using STTM technology.Through agrobacterium mediated and tissue culture,T₃ transgenic lettuce plants with stable inheritance were obtained.The results showed that the expression of miR394decreased and the expression of F-box increased in transgenic lettuce;We speculate that F-box may promote the expression of HSP70 and HSP90.As molecular chaperones,they participate in the regulation of lettuce response to high temperature stress,so as to improve the high temperature resistance of lettuce.