| Drought and high temperature are one of the most important environmental factors that seriously threaten the agriculture and animal husbandry production.With the intensification of human activities,the trend of global warming is intensifying,which has led to increased frequency and severity of extreme drought and high temperature events.Alfalfa(Medicago sativa L.)as an excellent legume forage,plays an important role in the agriculture and animal husbandry production.Northern China is the largest alfalfa-producing region.However,the contradiction between grain crops and forages competing for limited farmland resource in northern China is becoming more and more serious.Therefore,developing the alfalfa cultivars with improved drought and heat tolerance will be necessary to sustainable development of alfalfa productivity and utilization of marginal lands in northern China.Nevertheless,given alfalfa was cultivated under favorable conditions of water and nutrients for long time,its genetic potential to adapt to various adverse environments has been lost.Desert plants have gradually evolved complex and unique resistance mechanisms in the long-term adaptation to extreme environments,which provide abundant genetic resource for improving forage and crop.Zygophyllum xanthoxylum is a typical xerophyte that widely distributes in the deserts of northwest China and arid and semi-arid areas of Central Asia.Previous studies have shown that well-developed cuticle on the leaf surface is an important characteristic of Z.xanthoxylum to adapt to extreme habitat.Cuticular wax,as the most important component of the cuticle,which plays a key role in plant resistance to environmental stresses,particularly drought stress.In our previous work,ZxABCG11,a wax transporter gene,was cloned from Z.xanthoxylum and induced by salt,osmotic,heat,combined osmotic and heat stresses.In addition,overexpression of ZxABCG11 significantly improved the drought resistance of Arabidopsis thaliana.Therefore,the objectives of this studies are to analyse the function of ZxABCG11 in cuticular wax transport in Arabidopsis;and further improve the stress resistance of alfalfa by manipulating ZxABCG11.The main findings are as follows:1.Scanning electron microscopy and Nile red staining showed that the waxy crystals on stems surface of ZxABCG11 transgenic Arabidopsis were significantly increased compared with wild type.Transmission electron microscopy showed that the cuticle of the transgenic Arabidopsis leaves was significantly thickened,which were increased by49%,38%,83% and 74% in four transgenic lines(MA1,MA2,AA1 and AA2)compared with the wild type,respectively.These results indicated that the overexpression of ZxABCG11 contributes to the formation of well-developed cuticle in Arabidopsis.2.Compared to wild type,the water loss and chlorophyll leaching rate of the transgenic Arabidopsis leaves were significantly lower.In addition,the cuticular wax content of transgenic Arabidopsis leaves and stems increased by 30%-46% and 36%-54% than those of wild type under normal conditions,respectively;under drought condition,it increased by 31%-54% and 44%-62%,respectively.RT-q PCR analysis showed that the expression abundance of At CER1,At CER2,At CER3,At CER4,At KCR1 and At LTPG1 in transgenic plants leaves and stems were significantly up-regulated.The expression level of At MAH1 that involved in the synthesis of secondary alcohol and ketones was significantly increased in stems,but there is not changes in leaves.These results suggesting that the overexpression of ZxABCG11 results in the accumulation of cuticular wax and the reduction of cuticular permeability,thereby increasing the drought tolerance.3.PCR and RT-PCR were used to detect the transgenic alfalfa.The results showed that ZxABCG11 from Z.xanthoxylum was successfully transformed into “Xinjiang Daye” alfalfa,and identified two lines with the higher expression levels(OE2 and OE9).The growth status of transgenic alfalfa was analyzed,we found that the growth performance of the two transgenic lines was significantly better than that of wild type,and plant height,leaf thickness and stem diameter were significantly increased relative to wild type,indicating that overexpression of ZxABCG11 was beneficial to the growth of transgenic alfalfa.4.Nile red staining showed that the waxy crystals of transgenic alfalfa leaf were significantly increased,and the cuticle thickness of OE2 and OE9 lines was 3.4-and3.3-fold compared with wild type,respectively.Gas chromatography-mass spectrometry analysis showed that the wax content of OE2 and OE9 lines leaves increased by 57% and 62% compared with wild type,respectively.RT-q PCR showed that the expression levels of Ms CER1,Ms CER2,Ms CER4 and Ms LTPG1 were significantly up-regulated in transgenic alfalfa leaves,indicating that ZxABCG11 positively regulates the biosynthesis and transport of cuticular wax,thereby increasing the cuticular wax content of transgenic alfalfa.5.Under either drought(30% field water capacity)or heat(40°C)treatment,the plant height,shoot dry and fresh weight,root dry and fresh weight,leaf area of transgenic alfalfa were significantly higher than those of wild type.In addition,the net photosynthetic rate,chlorophyll content and water use efficiency of transgenic lines leaves were significantly increased.After drought or heat treatment,compared to wild type,the wax content of the two transgenic lines was increased by approximately 54%and 48%,respectively;and the leaf water loss and chlorophyll leaching rate were obviously decreased.6.Under field conditions of hot arid region,the plant height,shoot dry and fresh weight,leaf area,stem diameter,chlorophyll a content,chlorophyll b content of OE2 and OE9 lines were significantly higher than those of wild type,and hay yield was increased by 56% and 50% compared with wild type,respectively.This study indicated that ZxABCG11 from Z.xanthoxylum plays an important role in plant response to drought stress.In addition,we systematically analyzed drought tolerance,heat tolerance and field performance of transgenic alfalfa,which lays a foundation for breeding new varieties of alfalfa with stress resistance and high yield,and provides an effective avenue for improvement of forage and crops by introducing excellent stress tolerance genes from desert plants. |
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