苹果cystatin家族基因MpCYS4在低氮胁迫响答中的功能研究

发布时间：2018-04-07 17:35

本文选题：M26　切入点：低氮胁迫　出处：《西北农林科技大学》2017年硕士论文

【摘要】：植物半胱氨酸蛋白酶抑制剂(CYS)是在植物体内能可逆地抑制半胱氨酸蛋白酶活性的一类蛋白家族。CYS基因已在拟南芥、水稻、大豆等多种植物中被鉴定,并揭示了该类基因在不同物种中的结构特征和生物学功能。苹果属于喜硝植物,氮胁迫会严重影响苹果的生长发育、果实品质和产量。有关苹果CYS基因在氮胁迫下的功能研究,尚未见报道。本课题前期鉴定了苹果cystatins基因家族成员,在富平楸子中克隆出MpCYS4基因(即Md CYS21),并获得过表达MpCYS4基因的M26转基因植株。本研究在此基础上,系统的分析了苹果CYS基因在低氮胁迫下的表达的变化。采用组培、沙培和水培的实验手段研究了MpCYS4转基因植株对低氮胁迫的抗性。获得如下结果:1、苹果砧木M26低氮胁迫处理的结果表明:低氮处理显著抑制了M26的生长。低氮处理组M26的干重、株高、茎粗、叶绿素含量、净光合速率等指标显著小于对照组,植株表现出典型的缺氮症状。在课题前期研究基础上,选取6个苹果CYS基因,分析其在低氮胁迫下的表达发现,这6个基因均受低氮胁迫的诱导,表达量上调显著。初步确认选取的6个苹果CYS基因(Md CYS1、Md CYS8、Md CYS15、Md CYS20、Md CYS21、Md CYS26)均参与了M26低氮响应过程。2、组培法研究MpCYS4基因响应低氮胁迫的结果表明:极低氮处理和无氮处理,转基因植株生长势优于野生型。并且,转基因植株的根系的根的数目、长度、表面积等方面明显大于野生型。初步表明MpCYS4基因的过量表达增强了M26对氮胁迫的抗性。3、沙培法研究MpCYS4基因响应低氮胁迫的结果表明:低氮条件下,M26转基因植株的根部和地上部的生物量、光合色素含量和净光合速率明显大于野生型,但是根冠比小于野生型。同时,MpCYS4基因的过量表达,导致低氮胁迫下叶片的气孔导度下降,叶片中H2O2积累量显著降低,而且成熟叶片中全氮含量大于野生型。4、水培法研究MpCYS4基因响应低氮胁迫的结果表明:低氮条件下,M26转基因植株的根部和地上部的生物量、光合色素含量明显大于野生型。M26转基因植株中超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)的活性显著大于野生型,叶片中的H2O2积累量显著低于野生型植株。M26转基因植株叶片中硝酸根转运蛋白基因Md NPF6.8、Md NPF6.9、Md NRT2.4和Md NRT2.7的表达量上调显著。综上所述,通过低氮胁迫下M26的表型与苹果CYS基因表达,及过表达MpCYS4转基因M26植株对低氮胁迫的抗性的关联分析,初步确定苹果CYS基因参与了植物的低氮响应。
[Abstract]:CYS, a plant cysteine protease inhibitor, is a family of proteins that can inhibit the activity of cysteine protease in plants. CYS gene has been identified in Arabidopsis, rice, soybean and many other plants.The structural characteristics and biological functions of these genes in different species were revealed.Apple is a nitrate plant, nitrogen stress will seriously affect the growth and development of apple, fruit quality and yield.The function of CYS gene in apple under nitrogen stress has not been reported.In this study, the members of cystatins gene family of apple were identified, MpCYS4 gene (Md CYS21) was cloned from Liriodendron mandshurica, and M26 transgenic plants expressing MpCYS4 gene were obtained.On the basis of this study, the changes of CYS gene expression in apple under low nitrogen stress were systematically analyzed.The resistance of MpCYS4 transgenic plants to low nitrogen stress was studied by tissue culture, sand culture and hydroponics.The results were as follows: 1. The results of low nitrogen stress on apple rootstock M26 showed that the growth of M26 was significantly inhibited by low nitrogen treatment.The dry weight, plant height, stem diameter, chlorophyll content and net photosynthetic rate of M26 in low nitrogen treatment group were significantly lower than those in control group.On the basis of previous study, six apple CYS genes were selected and their expression under low nitrogen stress was analyzed. It was found that all of the six genes were induced by low nitrogen stress and the expression levels were significantly up-regulated.The growth potential of transgenic plants was better than that of wild type.Moreover, the number, length and surface area of roots of transgenic plants were obviously larger than that of wild type.The results showed that the overexpression of MpCYS4 gene enhanced the resistance of M26 to nitrogen stress. The results of MpCYS4 gene response to low nitrogen stress showed that the biomass of root and shoot of M26 transgenic plants under low nitrogen stress was increased.The photosynthetic pigment content and net photosynthetic rate were significantly higher than those of wild type, but the ratio of root to shoot was lower than that of wild type.At the same time, the overexpression of MpCYS4 gene resulted in the decrease of stomatal conductance and H2O2 accumulation in leaves under low nitrogen stress.The total nitrogen content in mature leaves was higher than that in wild type. The results of MpCYS4 gene response to low nitrogen stress showed that: under low nitrogen condition, the biomass of root and shoot of M26 transgenic plants was higher than that of wild type.The content of photosynthetic pigment was significantly higher than that of wild type. M26 transgenic plants. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) were significantly higher than those of wild type.The accumulation of H2O2 in leaves was significantly lower than that in wild-type plants. Md NPF6.8 and Md NPF6.9 Md NRT2.4 and Md NRT2.7 were up-regulated significantly.In conclusion, the relationship between the phenotype of M26 under low nitrogen stress and the expression of CYS gene in apple and the resistance to low nitrogen stress of transgenic M26 transgenic plants with overexpression of MpCYS4 was analyzed. It was preliminarily confirmed that the CYS gene of apple was involved in the response to low nitrogen stress in plants.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S661.1

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