| High temperature,drought and other disasters caused by global war ming have led to a large number of reduction in grain yields.which has become an important factor restricting global crop yield and economic development.Plant surface wax is a layer of hydrophobic protective film in direct contact with the external environment,which can mobilize the water in the plant to resist drought,high temperature and other abiotic threats,so it plays an important role in the growth and development of the plant.Karelinia caspia has the characteristics of salt and alkali resistance,drought resistance,high temperature resistance and sand burial resistance.The epidermal layer is also thick with wax,which can play a key role in plant resistance to high temperature,drought and other abiotic stresses.Cotton is an important cash crop in China.Its growth and development are severely affected by abiotic stress such as salinity,moisture and extreme high temperature,which are important reasons to restrict cotton yield.In this study,differentially expressed genes were selected through the wax synthesis pathway of high-temperature expression spectrum of Karelinia caspia,and the wax synthesis related genes KcFAD2 and KcHHT were cloned for subsequent experiments.Subcellular localization vectors of KcFAD2 and KcHHT were constructed and transformed into transient expression in tobacco leaves.Confocal microscopy results showed that the green fluorescence of KcFAD2 was mainly on the cell membrane and co-located with the red fluorescence of the membrane Marker,indicating that KcFAD2 protein was located on the cell membrane.The green fluorescence of KcHHT is mainly located in the nucleus and cell membrane,which can co-locate with the red fluorescence of nuclear membrane Marker,indicating that KcHHT protein is located in the nucleus and cell membrane.Two gene overexpression vectors were constructed and transferred into tobacco and cotton by agrobacterium-mediated method.The positive test results showed that 12 KcFAD2-positive transgenic tobacco and 10 KcHHT-positive transgenic tobacco were obtained,and KcFAD2 and KcHHT were overexpressed in cotton.We obtained 20 KcFAD2-positive transgenic embryoids and 2 transgenic cotton seedlings,18 KcHHT positive transgenic embryoids and 1 transgenic cotton seedling,which provided new strategies and important resources for subsequent cotton stress resistance.The expression levels of KcFAD2 and KcHHT genes in transgenic materials were detected and all of them were up-regulated.Plants with high expression levels were selected for high-temperature treatment experiment.10 h 42 ? high temperature processing results show that KcFAD2 transgenic tobacco plants were more heat-resistant than control plants,the activity of superoxide dismutase(SOD),content of proline and soluble sugar content were significantly higher than that of control plant,malondialdehyde content was partly lower than control plants,Moveover,the wax content high temperature processing and significant correlation;KcHHT transgenic tobacco is more tolerant to heat sterss,the activity of superoxide dismutase(SOD),content of proline are significantly higher than the negative control plants,the content of soluble sugar were significantly higher than that of control plant,the content of malondialdehyde part was significantly lower than control plants,wax content after high temperature treatment were rising and significant correlation.These results indicated that the overexpression of KcFAD2 and KcHHT genes could improve the high temperature tolerance of plants.Meanwhile,the comparative analysis of drought expression profiles of drought-resistant cotton line ZY7 and drought-sensitive cotton line ZY168 also showed that KcFAD2 and KcHHT genes played a certain role in promoting stress tolerance.It has reference value for the mining and utilization of desert plant stress resistance gene resources. |
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