摘要
小麦作为世界上最重要的粮食作物之一，其产量关系着国家粮食安全和国民经济的发展。由大麦黄矮病毒(Barley yellow dwarf viruses，BYDVs)引发的小麦黄矮病，是我国小麦种植区最重要的小麦病毒病害之一，同时也是世界上危害最严重、流行最广泛的植物病毒病害之一。该病毒流行年份一般会造成20％～30％的减产，严重时达50％以上，甚至颗粒无收，被称为小麦的“黄色瘟疫”和“癌症”。由小麦矮缩病毒（Wheat dwarf virus，WDV）引发的小麦矮缩病在欧洲、北非、亚洲和大洋洲造成了严重的经济损失，特别是在欧洲该病害造成小麦减产40%～80%。2007年本实验室在国内首次报道了小麦矮缩病的发生，随后该病害在陕西、甘肃、河北、云南等12个省陆续被报道发现，尤其在陕西北部麦区已经引起小麦严重的减产，成为威胁我国西北、华北和西南麦区重要的病毒病害。
防治与控制病毒病最为经济有效的方法是利用品种自身的抗性从而达到主动防治病害的目的，然而利用传统的育种方式很难获得兼抗两种病毒病和高产、优质品种。RNA干涉（RNA
interference，RNAi）是指由双链RNA的介导，特异性靶标对应序列的mRNA，从而抑制相应基因表达的一种基因表达与调控方式，也是寄主一种重要防御病毒病侵害的机制。当表达来源于病毒某一基因序列的转基因植物发生RNAi时，就能使入侵的同一病毒或同属中相近病毒的RNA降解，从而赋予转基因植物对病毒的抗性。因此利用RNAi获得抗BYDVs和WDV小麦种质，成为解决这两种病害的有效途径。
随着转基因植物研究的深入和转基因作物的大范围种植，转基因生物安全性评价标准也在不断提高，人们期望获得无筛选标记基因和其他载体骨架的转基因新种质，这也成为目前转基因植物研究的热点之一。
本研究针对BYDV-GAV和WDV的CP基因的高保守性，构建了RNAi植物表达载体和最小表达框，期望利用基因枪介导的遗传转化和最小表达框技术，筛选获得高抗或免疫的小麦种质。为最终实现利用RNAi获得抗BYDVs和WDV小麦新品种的预期目标提供抗病新种质。主要结果如下：
1.表达载体的构建。成功的构建成了适用于小麦基因枪转化的载体：双价表达载体有pMCG161+/-BW、pMCG161+BW、pMCG161-BW；pWMB006+/-HBW、pWMB006+HBW、pWMB006-HBW。单价表达载体有pMCG161+/-W、pMCG161+W、pMCG161-W。
另外通过酶切获得+/-HBW最小表达框。
2.基因枪转化小麦及转化后代的分子检测。将构建好的载体和最小表达框利用基因枪法转化小麦幼胚幼胚愈伤组织，经过筛选和抗性愈伤的分化得到再生植株，再通过PCR检测获得了pMCG161+/-BW阳性植株36株，pMCG161+BW和pMCG161-BW均获得5株；pWMB006+/-HBW获得20株，pWMB006+HBW和pWMB006-HBW均获得5株；pMCG161+/-W获得10株，pMCG161+W和pMCG161-W均获得4株；转最小表达框的获得11株。平均转化率是1.2%，最高转化率是2.0%。Southen Blotting结果显示：在T3代稳定遗传的3个转基因株系中外源基因已经成功整合到小麦基因组染色体上，拷贝数为1-3个。对T3代稳定遗传的转基因株系中取8个株系，进行了实时荧光定量PCR检测，结果表明：转基因植株中目的基因的表达量低于受体材料，pMCG161+/-BW的一个株系P7-5-2相对基因表达量为0.3；P11-1-3的相对基因表达量为0.24；转pWMB006+/-HBW载体植株6个株系中5个株系的目的基因干扰效果很明显，其相对基因表达量0.15。
3．转基因植株的抗病鉴定。对稳定遗传的株系在T3代和T4代植株进行了BYDV-GAV的抗病性鉴定，获得了免疫株系有：14个转干扰载体pMCG161+/-BW的株系，9个独立转化事件；5个转干扰载体pWMB006+/-HBW的株系，5个独立转化事件；后者有望筛选到无标记基因的转基因抗病种质，为抗病育种提供新材料。
Wheat is one of the most important food crop in the world and plays a vital role in food securityand national economy. Wheat yellow dwarf disease caused by Barley yellow dwarf virus (BYDVs) isone of the most destructive viral disease of wheat crop not only in China but also in the world. Theepidemics of BYDVs generally leads to a yield loss around20%to30%, even up to50%or completeproduction failure compared to the normal production level.
In recent year, wheat dwarf disease caused by Wheat dwarf virus (WDV) has been an increasingdestructive viral disease in Europe, North Africa, Asia and Oceania. Especially in Europe, the yield losscaused by WDV has been over40%to80%. Wheat dwarf disease disease was firstly detected andreported by our research group in2007in China. Subsequently, it was found in12provinces includingShaanxi, Gansu, Hebei and Yunnan. This disease, which caused serious yield loss in Hancheng city,north of Shaanxi province, has been a potential threat to the wheat in northwestern of China.
The most efficient and economic method to control viral disease is the application of resistantvarities. However, it is very hard to obtain a new resistant wheat varity with high yield and goodquantity through traditional breeding. The recently discovered RNA interference (RNAi) is an importantmechanism of host against pathogens by specific degradation of the mRNA mediated bydouble-stranded RNA. The transgenic plant could show the resistance to the viral disease when the virusgenes or their homologous fragment were degraded by RNA interference. It gives us a opportunity thatwheat lines with resistance to BYDVs and WDV could be obtained by transgenic breeding based on theprinciple of RNAi.
The purpose of this research is to obtain the transgenic wheat lines with high resistance toBYDV-GAV and WDV. Firstly, we developed a series of RNAi vectors containing conserved coatprotein gene sequences of BYDV-GAV and WDV, which are suitable for wheat transformation. Theseplasmid constructs were transformed into callus of wheat immature embryo by particle bombardment.The genetically modified regeneration were obtained after the tissue culture. The seedlings were testedand selected by PCR, Southen blotting, Q-RT-PCR and bioassay. In addition, the linear minimumexpression box for particle bombardment transformation was designed for biosafety of transgenic wheat.The expression box was transformed into callus of wheat immature embryo via particle bombardmentand the positive seedlings were obtained by PCR. Among of them, the resistance plants without othervector sequence and antibiotic gene could be screened for the next application. The main results arelisted as follows:
1. Construction of RNAi expression vector: We have developed RNAi expression vectors for wheattransformation including pMCG161+/-BW, pMCG161+BW, pMCG161-BW, pMCG161+/-W,pMCG161+W, pMCG161+/-W, pMCG161-W, pWMB006+/-HBW, pWMB006+HBW,pWMB006-HBW and gene expression casseett+/-HBW.
2. Biolistic transformation and molecular detection of foreign genes: Those vectors had beentransfered into immature wheat embryos. Then transgenic tissues were selected by herbicide resistance and detected by molecular methods. PCR results showed36plants positive for pMCG161+/-BW,5plants positive for pMCG161+BW,5plants positive for pMCG161-BW,20plants positive forpWMB006+/-HBW,5plants positive for pWMB006+HBW,5plants positive for pWMB006-HBW,10plants positive for pMCG161+/-W,4plants positive for pMCG161+W,4plants positive forpMCG161-W,11plants positive for gene expression casseett+/-HBW. respectively. The averagetransformation rate and the highest transformation rate were1.2%and2.0%respectively. Southenblotting analysis showed that the foreign sequence had been integrated into the transgenic wheatgenome with1-3copies. Real time quantitative PCR analysis revealed that the expression levels of thetarget genes in transgenic plants was relatively lower than them in wild type. The gene expression ofpositive plants which transformed with pMCG161+/-BW1is0.3of the controled ones, withpMCG161+/-BW2is0.24and with pMCG161+/-HBW is0.15. It indicated that the gene silencingeffect played a role in transgenic plants.
3. Bioassay of transgenic plants: By observation of their agronomic characters of T1plant, they werethe same as controls. The evaluation of the lines T2and T3resistant to BYDVs by artificial inoculationshowed that disease resistant GM wheats had been obtained.
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