荔枝凝集素基因的克隆与表达分析
摘要
荔枝是一种热带名优水果,在我国热带农业经济中具有重要地位,但生产上面临病虫害和采后不耐储藏的困扰。凝集素是指含有至少一个催化结构域,并能与特异性单糖或寡糖进行可逆结合的蛋白质,这种糖识别特性使其能够提高植物的抗病虫能力。荔枝凝集素基因的克隆及实时表达研究对促进荔枝生产必将具有一定指导意义。
本文以妃子笑、南岛无核荔和紫娘喜为研究对象,首先对荔枝果实采后衰老过程中若干生理变化进行初步研究,同时运用PCR等方法克隆荔枝凝集素基因的编码区序列和启动子调控序列,并对序列进行生物信息学分析,在此基础上应用荧光定量PCR技术对凝集素基因在荔枝中的表达情况进行相对定量。结果如下:
1)荔枝果实采后衰老过程中若干生理变化
荔枝采后随着果皮褐变程度的加剧,果皮花色素苷含量减少,通透性增大,pH值增高,果实发病率上升。低温和套袋可以降低荔枝果皮水分的散失率,延缓荔枝褐变的进程。
2)荔枝凝集素基因的克隆与表达
从荔枝中克隆获得了荔枝凝集素基因cDNA和gDNA,其中cDNA序列全长为874bp,含有一个468bp的完整开放阅读框,所编码蛋白与木菠萝家族凝集素有着相似的结构域;克隆获得了荔枝凝集素基因5’上游约1300bp的启动子序列,该序列富含TATA框等启动子活性元件的基本特征;成功构建植物表达载体pCAMBIA1302-lectin。
3) lectin基因表达的相对定量
凝集素基因具有显著的部位表达特异性,其在茎和种子中的表达量远高于果肉;在果实动态发育过程中,凝集素基因在发育前期的表达量略高于中后期;果实采后衰老阶段,凝集素出现短时间内表达急剧升高的特点。本研究创新之处:
1)首次克隆获得荔枝凝集素基因cDNA和gDNA,并证实荔枝凝集素是由多基因家族所编码;
2)成功克隆获得了荔枝凝集素基因5’上游约1300bp的启动子序列,并构建了植物表达载体pCAMBIA1302-lectin,为凝集素蛋白的亚细胞定位和转基因研究提供了理论基础。
Litchi is a famous tropical fruit, which has an important position in the tropical agricultural economy of China. But it also faces with the problem that pests and diseases undermine litch fruit at growing and harvest storing. Lectin is a special protein which has one or more catalytic domain, which could distinguish and bind with monosaccharide or oligosaccharides of cell surface. Thus, lectin can improve resistance of plants to diseases and pests. It is necessary to intensive study expression of lectin gene in litch to improve litchi productivity.
Using Feizixiao, Nadaowuhe and Ziniangxi, difference of physiological characteristics changes was first investigated during storage. Lectin gene coding sequences and promoter regulatory sequence was then cloned and sequenced. On this basis of sequence, the relative quantity of lectin gene expression in the litchi was detected using quantitative PCR. Results are as follows:
1) During storage, with the increased of pericarp browning degree, anthocyanin content decreased, the permeability increased, pH value increased, and incidence of a disease increased. Low temperature and bagging can delay the process of browning by reducing water loss rate of litchi fruit.
2) Cloned the sequences of lectin gene cDNA and gDNA from litch. The cDNA full length sequence is 874bp, containing a complete open reading frame which seqence length is 468bp. The encoded protein has similar domain with jackfruit family lectin. A1300bp promoter sequences upper lectin gene 5’stream was cloned, which is rich of TATA box that often appear in Promoter’s Active Components. A plant expression vector“pCAMBIA1302-lectin”was also successfully constructed.
3) RT-PCR study showed that the expression of lectin gene is specific significant, the expression levels in stem and seeds are much higher than that in pulp. During growth and development of litch fruit, the expression level of lectin gene during early development slightly higher than the late. At senescence stage, the expression level of lectin gene showed a sharp increase with a short time.
We summarize the important novel findings as follows:
1) For the first time, successfully cloned the sequences of lectin gene from litch, and confirmed that litchi lectin was encoded by multiple gene family.
2) A1300bp promoter sequences upper lectin gene 5’stream was cloned, and a plant expression vector“pCAMBIA1302-lectin”was also successfully constructed for the first time, which provide a basis for studying the subcellular localization of litch lectin and transgenosis.
本文以妃子笑、南岛无核荔和紫娘喜为研究对象,首先对荔枝果实采后衰老过程中若干生理变化进行初步研究,同时运用PCR等方法克隆荔枝凝集素基因的编码区序列和启动子调控序列,并对序列进行生物信息学分析,在此基础上应用荧光定量PCR技术对凝集素基因在荔枝中的表达情况进行相对定量。结果如下:
1)荔枝果实采后衰老过程中若干生理变化
荔枝采后随着果皮褐变程度的加剧,果皮花色素苷含量减少,通透性增大,pH值增高,果实发病率上升。低温和套袋可以降低荔枝果皮水分的散失率,延缓荔枝褐变的进程。
2)荔枝凝集素基因的克隆与表达
从荔枝中克隆获得了荔枝凝集素基因cDNA和gDNA,其中cDNA序列全长为874bp,含有一个468bp的完整开放阅读框,所编码蛋白与木菠萝家族凝集素有着相似的结构域;克隆获得了荔枝凝集素基因5’上游约1300bp的启动子序列,该序列富含TATA框等启动子活性元件的基本特征;成功构建植物表达载体pCAMBIA1302-lectin。
3) lectin基因表达的相对定量
凝集素基因具有显著的部位表达特异性,其在茎和种子中的表达量远高于果肉;在果实动态发育过程中,凝集素基因在发育前期的表达量略高于中后期;果实采后衰老阶段,凝集素出现短时间内表达急剧升高的特点。本研究创新之处:
1)首次克隆获得荔枝凝集素基因cDNA和gDNA,并证实荔枝凝集素是由多基因家族所编码;
2)成功克隆获得了荔枝凝集素基因5’上游约1300bp的启动子序列,并构建了植物表达载体pCAMBIA1302-lectin,为凝集素蛋白的亚细胞定位和转基因研究提供了理论基础。
Litchi is a famous tropical fruit, which has an important position in the tropical agricultural economy of China. But it also faces with the problem that pests and diseases undermine litch fruit at growing and harvest storing. Lectin is a special protein which has one or more catalytic domain, which could distinguish and bind with monosaccharide or oligosaccharides of cell surface. Thus, lectin can improve resistance of plants to diseases and pests. It is necessary to intensive study expression of lectin gene in litch to improve litchi productivity.
Using Feizixiao, Nadaowuhe and Ziniangxi, difference of physiological characteristics changes was first investigated during storage. Lectin gene coding sequences and promoter regulatory sequence was then cloned and sequenced. On this basis of sequence, the relative quantity of lectin gene expression in the litchi was detected using quantitative PCR. Results are as follows:
1) During storage, with the increased of pericarp browning degree, anthocyanin content decreased, the permeability increased, pH value increased, and incidence of a disease increased. Low temperature and bagging can delay the process of browning by reducing water loss rate of litchi fruit.
2) Cloned the sequences of lectin gene cDNA and gDNA from litch. The cDNA full length sequence is 874bp, containing a complete open reading frame which seqence length is 468bp. The encoded protein has similar domain with jackfruit family lectin. A1300bp promoter sequences upper lectin gene 5’stream was cloned, which is rich of TATA box that often appear in Promoter’s Active Components. A plant expression vector“pCAMBIA1302-lectin”was also successfully constructed.
3) RT-PCR study showed that the expression of lectin gene is specific significant, the expression levels in stem and seeds are much higher than that in pulp. During growth and development of litch fruit, the expression level of lectin gene during early development slightly higher than the late. At senescence stage, the expression level of lectin gene showed a sharp increase with a short time.
We summarize the important novel findings as follows:
1) For the first time, successfully cloned the sequences of lectin gene from litch, and confirmed that litchi lectin was encoded by multiple gene family.
2) A1300bp promoter sequences upper lectin gene 5’stream was cloned, and a plant expression vector“pCAMBIA1302-lectin”was also successfully constructed for the first time, which provide a basis for studying the subcellular localization of litch lectin and transgenosis.
引文
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