木薯UGT85K4基因在培育抗旱和低氰木薯中功能的研究

Cassava is one of the most drought-tolerant crops and is an important economic crop in the tropical and subtropical regions.95% of the cassava cyanide was linamarin that was synthesized by CYP79D1, CYP71E7 and UGT85K4 protein.There was a positive correlation between cyanide content and drought resistance of Cassava Varieties.The cyanide content of cassava in drought stress was higher than that in normal water. Our study found MeUGT85K4 gene expression and UGT85K4 protein abundance were significantly raised in leaf and root of cassava under drought and the changes of CYP79D1 and CYP71E7 protein were not significant. Therefore, MeUGT85K4 gene might play a role in breeding drought resistant. We hypothesized that the UGT85K4 gene could increase the UGT85K4 protein in response to drought stress, and the UGT85K4 protein could catalyze more substrates to linamarin and enhance the drought resistance of cassava. This project used 35S promoter to drive MeUGT85K4 expression in cassava by transgenic technology mediated by Agrobacterium.tumefacienes, at the same time, RNAi technology was used to silence MeUGT85K4 gene. Whether MeUGT85K4 overexpression enhance and whether MeUGT85K4 silence reduce the drought resistance of cassava.It help us to study functional of MeUGT85K4 gene in drought-resistant of cassava. We study drought resistance mechanism of MeUGT85K4 by comparing its gene and protein quantity, linamarin and.nitrogen concentration in different organs of transgenic and contrast plants under drought. Linamarin was toxic to human and animal. MeUGT85K4 was indispensable in the synthesis of linamarin. To assess MeUGT85K4 gene role in cultivating low cyanide cassava, we compared linamarin concentration in mature root of interference MeUGT85K4 gene transgenic and contrast cassava plants .It was important to cultivate low cyanide cassava and to study drought resistance mechanism of UGT85K4 gene to promote development of cassava industry.

木薯是非常抗旱的作物，是热带亚热带地区重要的经济作物。UGT85K4 、CYP79D1和CYP71E7蛋白协同作用合成占木薯总氰化物含量95%的亚麻苦苷。有报道显示氰化物含量与木薯品种的抗旱性呈正相关性，并在干旱胁迫的木薯中升高。我们前期研究发现UGT85K4基因和蛋白响应干旱胁迫，在木薯叶和根中显著上调表达，但CYP79D1和CYP71E7蛋白的变化并不显著。我们推测UGT85K4基因响应干旱上调导致UGT85K4蛋白增加，UGT85K4蛋白催化更多的底物转化为亚麻苦苷，增强了木薯的抗旱性。本项目拟利用35S和RNAi技术控制MeUGT85K4基因的表达，比较干旱后木薯抗旱性、MeUGT85K4基因表达量、蛋白丰度和氰化物含量在转基因和对照木薯植株中的差异，研究MeUGT85K4基因的抗旱功能和机理。鉴于木薯中的氰化物对人畜有毒，本项目的结果可能为低氰木薯新种质的培育提供重要材料。

木薯是非常抗旱的作物，是热带亚热带地区重要的经济作物。我们前期研究发现UGT85K4基因和蛋白响应干旱胁迫，在木薯叶和根中显著上调表达。推测UGT85K4基因响应干旱上调导致UGT85K4蛋白增加，UGT85K4蛋白催化更多的底物转化为亚麻苦苷，增强木薯的抗旱性。本项目克隆了MeUGT85K4基因，对其表达模式和功能进行了研究，通过定量PCR发现其对PEG，ABA处理及干旱都有响应，呈上调表达。通过酵母双杂交试验筛选到在干旱和低温胁迫处理时与MeUGT85K4蛋白互作的候选蛋白6个，其中三个蛋白在干旱处理的木薯中差异表达，我们推测这些互作蛋白和MeUGT85K4蛋白协同作用参与木薯的抗旱应答。我们构建了过量表达（1个）和干扰表达（2个）MeUGT85K4基因的表达载体，通过农杆菌介导的的方法获得以C3脆性愈伤为材料的过量表达MeUGT85K4基因的转基因木薯3个株系（定量PCR检测MeUGT85K4基因高表达），以SC8脆性愈伤为材料的MeUGT85K4基因敲除木薯15个株系。通过对亚麻苦苷的测定显示过量表达的株系亚麻苦苷增加，敲除的株系亚麻苦苷降低。过量表达MeUGT85K4基因的转基因木薯组培苗和对照相比变得矮小。敲除MeUGT85K4基因的转基因木薯没有明显的表型特征。本项目将为低氰木薯、抗旱木薯新种质的培育提供重要材料。