芽孢杆菌植酸酶phyC基因对桑树的遗传转化研究

Environmental protection is an important task of ecological environment construction. For phosphorus pollution of environment, the pollution of cultivation industry is more serious than crop farming, the most important reason is the phosphorus exists in the form of phytic acid in plants, and cannot be utilized effectively by maongastric animals but will only be excreted outside, polluting water as well as the whole environment. In the previous study we found that the mulberry has abnormal high content phytic acid, and it also cannot be utilized effectively by silkworm. It is of great scientific significance to do research on reducing the phytic acid content of mulberry leaves using phyC gene. The project group had carryied out genetic transformation experiments with agrobacterium tumefaciens mediated method and pollen tube pathway method respectively, the results showed that using those two methods for transformation the phyC gene into mulberry is feasible and effective. This study intends to solve the problem of gene silencing by cloning the efficient tissue specific promoter from the own genome of mulberry and optimize the target gene according to codon usage bias in mulberry and amino acid sequence of the neutral phytase, and then we will have obtained mulberry plants with phytase activity, so as to increase the utilization rate of organic phosphorus in silkworm and reduce environmental pollution. On this basis, the author will try to establish and improve mulberry genetic transformation systems by combining the two methods; this will have significant application in genetic improvement of quality of mulberry leaves. At the same time it can provide a scientific basis for improving the utilization of phosphorus in plant feedstuffs.

环境保护是生态环境建设的重要任务。从磷对环境的污染而言，养殖业较种植业的污染更为严重，其主要原因为植物中的磷主要以植酸磷的形式存在，单胃动物不能吸收利用植酸磷而向外排泄，污染环境。在前期的研究中发现桑叶植酸含量较高，且试验表明家蚕不能吸收利用，因此利用中性植酸酶phyC基因降低桑树植酸含量的研究具有重要科学意义。项目组开展根癌农杆菌介导浸染桑丛生芽和花粉管通道法的遗传转化实验表明两种方法将phyC基因转化入桑树均是可行的。本项研究拟通过从桑树基因组中克隆组织特异性启动子和利用桑树密码子偏好性优化目的基因等来解决基因沉默问题，获得具有植酸酶活性的桑树植株，提高家蚕对桑叶植酸磷的吸收利用减少对环境的污染，同时结合两种转化方法进行评估，建立一套较为完善的桑树遗传转化系统，为桑叶品质改良提供理论依据，同时也为提高植物性饲料中磷的利用提供科学依据。

动物体内的磷主要来自于植物，植物中的磷主要以植酸磷的形式存在，单胃动物不能吸收利用植酸磷而要向外排泄污染环境。项目组经过前期的研究发现桑叶中的植酸含量较高并随着桑叶的成熟其含量不断增加，且家蚕不能吸收利用植酸态的磷，而桑叶营养丰富是家蚕唯一的饲料，其成熟叶的pH接近中性（pH=6.1），鉴于此本项目从土壤中筛选芽孢杆菌并从中克隆获得中性植酸酶phyC基因，利用原核大肠杆菌和真核酵母表达系统对该基因进行表达并纯化该酶蛋白，对其进行酶活性质及功能分析研究，然后利用桑树密码子偏好性对该基因序列进行优化合成，采用抑制PCR技术扩增桑树肌动蛋白启动子并且构建表达载体在拟南芥中进行瞬时表达验证该启动子的启动活性。将桑树肌动蛋白启动子和优化的基因一起构建表达框并克隆进植物表达载体pCAMBIA-2300，分别利用农杆菌侵染桑树组培不定芽和Floral Dip法进行了桑树的遗传转化研究。结果显示转化苗先进行卡那霉素初筛后，PCR鉴定获得阳性植株，Southern blotting显示其单株基因组均是含有一个T-DNA拷贝的形式存在，通过RT-PCR检测发现具有Southern杂交信号的三个单株具有微弱的表达，进一步利用Nothern blotting鉴定但是未能检测到杂交信号。通过对遗传转化阳性植株的观察发现其叶片颜色明显加深，叶片变得粗糙肥厚，顶芽出现了畸形的现象，说明外源基因的表达对植株的生长具有一定的影响，对三个阳性植株叶片的植酸进行检测发现其含量分别为3.59、3.74和3.49mg/g，略低于对照的4.02、4.02和3.74mg/g。通过该项目的实施我们优化授粉条件克服了重庆菌核病爆发严重和云南省草坝镇气候干燥不利于杂交授粉后农杆菌侵染的缺点，完成了获得具有少量植酸酶表达活性的转化植株的目标，初步得到的结果表明通过桑树组培不定芽很难获得具有植酸酶活性的植株，仅能在前期检测到PCR阳性，而利用Floral Dip法能检测到拷贝数较低的阳性植株且外源基因有少量的表达，在后期的研究中可以采取发根农杆菌和诱导桑树二次开花等措施来提高转化效率。