黄瓜衰老过程中叶绿素降解相关酶基因cDNA片段的克隆与表达初步研究
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摘要
叶绿素(Chlorophyll, Chl)是绿色植物叶绿体内参与光合作用的重要色素,叶绿素降解是衰老的一个重要现象,叶绿素降解是植物自然生长的结果,也可能受衰老诱导；植物衰老的一个显著标志是失绿。本文以衰老表现不同的两种黄瓜,‘华黄5号,(Cucumis sativusL.var.Huahuang 5)和西双版纳黄瓜(Cucumis sativus L.var.xishuangbannanesis Qi etYuan)为试材,初步研究了野生种和栽培种衰老过程中生理生化特性,并且克隆叶绿素降解途径中的两个关键酶基因cDNA片段——脱镁叶绿素脱镁叶绿酸a水解酶(PPH)和脱镁叶绿酸a加氧酶(PAO)。应用Real-time PCR技术检测其在植株衰老过程中叶和茎中的相对表达量,探讨了PPH和PAO基因的表达模式及与抗衰老的关系,主要研究结果如下：
1.黄瓜叶片衰老过程中生理生化特性
黄瓜叶片衰老过程中,西双版纳黄瓜和‘华黄5号’黄瓜叶片叶绿素含量总体表现为先上升后下降。在叶片伸出15天时叶绿素含量最高,叶片伸出35天后,‘华黄5号’叶片开始出现老化症状,在45天时仅为15天的42%。而西双版纳叶片一直保持较高叶绿素含量,‘华黄5号’叶内SOD活性开始快速下降,后有微弱的上升,在35天后下降,而西双版纳黄瓜在35天时活性最高,后微弱下降,野生种SOD活性开始出现下降的时间比栽培种晚20天,可能在35天西双版纳黄瓜叶片才开始衰老。‘华黄5号’的POD活性一直呈上升趋势,而西双版纳黄瓜的POD活性变化呈双峰曲线模式,除在5天时活性低于‘华黄5号’,其余天数远远高于‘华黄5号’。西双版纳黄瓜和‘华黄5号’叶片POD活性在45天最高,在生长后期,野生种在清除活性氧方面仍强于栽培种。西双版纳黄瓜叶片中可溶性蛋白含量高于‘华黄5号’,西双版纳黄瓜在35天时蛋白含量最高,‘华黄5号’在45天时含量最高,并且可溶性蛋白质变化规律不明显。
2.黄瓜茎衰老过程中生理生化特性
黄瓜茎衰老过程中,西双版纳黄瓜茎在45天叶绿素含量最高,和叶片不同,西双版纳茎中叶绿素含量变化趋势不明显,茎叶绿素含量最高出现在45天,表明野生种茎可能还未进入衰老期。‘华黄5号’茎叶绿素含量在25天最高,茎中叶绿素含量先上升后下降；野生种茎中叶绿素含量高于栽培种。在栽培种和野生种茎里SOD活性变化均呈现双峰式变化趋势,西双版纳黄瓜和‘华黄5号’在35天时茎中的SOD活性最高。西双版纳黄瓜茎的POD活性一直呈上升趋势,‘华黄5号’的POD变化有呈现双峰趋势。在45天时,西双版纳黄瓜和‘华黄5号’的POD活性达到最大,并且西双版纳黄瓜茎中的POD活性始终高于‘华黄5号’。在35天时西双版纳和‘华黄5号’黄瓜茎中可溶性蛋白的含量最高,整体变化趋势是先升高后下降,在45天时最低。表明此时茎已经走向衰老。
3.脱镁叶绿素脱镁叶绿酸a水解酶(PPH)和脱镁叶绿酸a加氧酶酶(PAO)基因克隆
克隆的PPH和PAO基因碱基序列片段长度分别为265 bp和230 bp, BLAST核酸同源性在线分析表明,PPH片段与拟南芥(AT5G13800)、花椰菜(OL386R)、葡萄(XM002271131.1)和烟草(AM851012.1)的该家族基因分别具有87%、88%、80%和78%的核苷酸序列同源性,PAO片段与拟南芥(NM-114357.5)、葡萄(FJ799360.1)、花椰菜(AB470926)、毛果杨(XM002331446.1)和蓖麻(XM002523689)的该家族基因分别具有90%、86%、85%、83%和82%的核苷酸序列同源性,证明该扩增产物是PPH和PAO酶基因片段。
4.西双版纳和‘华黄5号’黄瓜叶衰老过程中PPH和PAO基因的相对表达量。
PPH和PAO基因在西双版纳黄瓜叶中的相对表达量的变化趋势有所不同,PPH基因的相对表达量是先上升后下降,在35天时表达量最高。PAO基因在15、25天时表达较弱,在35天时表达最强。PPH和PAO基因在‘华黄5号’叶的相对表达量与在西双版纳黄瓜叶片里表达模式基本相同,PPH基因的相对表达量是先上升后下降,在35天时表达量最高,PAO基因在15、25天时表达较弱,和在西双版纳不同的是,PAO基因在45天表达最强。这两个基因在西双版纳和‘华黄5号’叶片中表达呈正相关。
5.西双版纳和‘华黄5号’黄瓜茎衰老过程中PPH和PAO基因的相对表达量。
与在叶片中的表达模式相似,PPH和PAO基因在西双版纳黄瓜茎中的相对表达量的变化趋势也有所不同,PPH基因在茎中的表达趋势和在叶片中的表达趋势相同,先上升后下降,在35天时最强。PAO基因的表达变化趋势是先上升、后下降、再上升,在45天表达最强。和在西双版纳黄瓜茎里的表达模式不同,PPH和PAO基因在‘华黄5号’茎的表达模式是上升、稍微下降、上升、稍微下降。并且和5日的参照值相比都有一个较高的表达量。PPH在35天表达最强,PAO基因在15天表达最强。相关性分析表明,这两个基因在‘华黄5号’茎中表达呈正相关,而在西双版纳茎中表达呈负相关。
Chlorophyll is an important pigment in the plant chloroplasts being concerned with photosynthesis.Chlorophyll degradation is an important phenomenon in the senescence process. Chlorophyll is degraded in plants as a result of natural or induced senescence.Loss of green color is the most obvious sign of plant senescence.In this study, the cultivar Cucumis sativusL.var.Huahuang 5 and the wild species Cucumis sativus L.var.xishuangbannanesis Qi etYuan with different senescence were used as experient materials.The physiological and biochemical characteristics were preliminary studied during senescence in the wild species and the cultivar. Meanwhile, Two key enzymes gene of Chlorophyll degradation (Pheophytin Pheophorbide Hydrolase and Pheideaoxygenase) were cloned. Expression of these two genes during leaf and stem senescence was quantified through real-time PCR technique. The relationship between the expression model of PPH and PAO genes and anti-senescence were discussed. Major results are as follows:
1. The physiological and biochemical characteristics of cucumber leaf during senescence
As xishuangbannanesisQietYuan and Huahuang 5 became senescent, chlorophyll content in leaves increased first,but decreased later. The Chlorophyll content was the highest at 15d. The ageing symptoms of leaf in Huahuang 5 appeared at 35d, the contents at 45d were 45% at 15d. on the contrary, chlorophyll content in xishuangbannanesisQi etYuan was always a higer level. The activities of SOD in Huahuang 5 decreased dramatically first, but increased faint later, decreased at 35d again. The activities of SOD of xishuangbannanesisQi etYuan at 35d was the highest, decreased later, The activities of SOD in the wild species started to decrease 20 days later than the cultivar, Maybe the senescent of xishuangbannanesisQietYuan start at 35d.The activities of POD of Huahuang 5 all raise as cucumber became senescent, POD showed two peaks in the wild species, The activities of POD was higher than Huahuang 5 except at 5d. The activities of POD of Huahuang 5 and xishuangbannanesisQi etYuan was the highest at 45d. the ability of the wild species which cleaned out active oxygen was stonger than the cultivar at the later stage of cucumber growth. The soluble protein content of xishuangbannanesis Qi etYuan was higher than Huahuang 5, The Chlorophyll content was the highest at 35d. The soluble protein content of Huahuang 5 was at 45, meanwhile, the variation laws of the soluble protein was not significantly.
2.The physiological and biochemical characteristics of cucumber stem during senescence
As xishuangbannanesis QietYuan and Huahuang 5 became senescent, The Chlorophyll content in stem of xishuangbannanesisQi etYuan was the highest at 45d. unlike leaf, the variation trends of the Chlorophyll content in stem of xishuangbannanesis Qi etYuan was not significantly. Maybe the stem didn't enter the senescent.The Chlorophyll content in stem of Huahuang 5 was the highest at 25d. chlorophyll content in stems increased first, but decreased later.Thecontent of chl of xishuangbannanesisQi etYuan was higher than Huahuang 5.
The activities of SOD of xishuangbannanesisQi etYuan and Huahuang 5, showed two peaks. It were the highest at 35d.The activities of POD of xishuangbannanesis Qi etYuan all raise as cucumber became senescent, it showed two peaks in the cultivar, The activities of POD of Huahuan 5 and xishuangbannanesisQi etYuan was the highest at 45d. The activities of POD was higher than Huahuang 5.The soluble protein content of xishuangbannanesis Qi etYuan and Huahuang 5 were the highest at 35d, overall, the variation trends of the soluble protein was in stem increased first,but decreased later.the lowest were at 45d.
3. Cloning of PPH and PAO genes.
The length of cloned PPH and PAO genes were 265 base pairs and 230 base pairs, Analysis of the nucleotide sequenee by BLAST on line indicated that homology of PPH gene was up to 87% between Cucumis sativusL.var. and Arabidopsis thaliana (L.) Heynh, up to 88% between it and Brassica oleracea L. var. botrytis L, up to 80% between it and Vitis vinifera, up to 78% between it and Nicotiana tabacum. homology of PAO gene was up to 90% between Cucumis sativusL.var. and Arabidopsis thaliana (L.) Heynh, up to 86% between it and Brassica oleracea L. var. botrytis L, up to 85% between it and P. trichocarpa, up to 82% between it and Ricinus communis.
4. Relative quantification of PPH and PAO genes during leaf senescence of Cucumis sativus L.var.xishuangbannanesis Qi etYuan and Huahuang 5.
It is different that expression of both PPH and PAO genes in the leaf of xishuangbannanesisQi etYuan. The relative expression of PPH gene basically up-regulated first, down-regulated later. It was the highest expression at 35d. The minimum expression of PAO gene at 15d and 25d.The maximum at 35d. Expression of PPH and PAO genes between cultivars and wild species was quite similar. The relative expression of PPH gene basically up-regulated first,down-regulated later in stem of Huahuang 5. The maximum at 35d too, the minimum expression of PAO gene at 15d and 25d, the maximum at 45d. There was a positive correlation between both PPH and PAO genes in the leaf of xishuangbannanesis Qi etYuan and Huahuang 5.
5. Relative quantification of PPH and PAO genes during stem senescence of Cucumis sativus L.var.xishuangbannanesis Qi etYuan and Huahuang 5.
Unlike the expression modle of leaf. It is different that expression of both PPH and PAO genes in the stem of xishuangbannanesis Qi etYuan. Expression of PPH and PAO genes between leaf and stem was quite similar. The relative expression of PPH gene basically up-regulated first, down-regulated later too.it was the highest expression at 35d. The expression mold of PAO gene basically up-regulated first, down-regulated later, then up-regulated. The maximum at 45d. Unlike the expression modle of stem of xishuangbannanesis Qi etYuan, expression of both genes basically down-regulated first, up-regulated, down-regulated, up-regulated last, it showed two peaks in the cultivar,there was a higher expression than the value of at 5d. The maximum expression of PPH at 35d, PAO at 15d. There was a positive correlation between both PPH and PAO genes in the stem of Huahuang 5. negative correlation in the stem of xishuangbannanesis Qi etYuan.
1.黄瓜叶片衰老过程中生理生化特性
黄瓜叶片衰老过程中,西双版纳黄瓜和‘华黄5号’黄瓜叶片叶绿素含量总体表现为先上升后下降。在叶片伸出15天时叶绿素含量最高,叶片伸出35天后,‘华黄5号’叶片开始出现老化症状,在45天时仅为15天的42%。而西双版纳叶片一直保持较高叶绿素含量,‘华黄5号’叶内SOD活性开始快速下降,后有微弱的上升,在35天后下降,而西双版纳黄瓜在35天时活性最高,后微弱下降,野生种SOD活性开始出现下降的时间比栽培种晚20天,可能在35天西双版纳黄瓜叶片才开始衰老。‘华黄5号’的POD活性一直呈上升趋势,而西双版纳黄瓜的POD活性变化呈双峰曲线模式,除在5天时活性低于‘华黄5号’,其余天数远远高于‘华黄5号’。西双版纳黄瓜和‘华黄5号’叶片POD活性在45天最高,在生长后期,野生种在清除活性氧方面仍强于栽培种。西双版纳黄瓜叶片中可溶性蛋白含量高于‘华黄5号’,西双版纳黄瓜在35天时蛋白含量最高,‘华黄5号’在45天时含量最高,并且可溶性蛋白质变化规律不明显。
2.黄瓜茎衰老过程中生理生化特性
黄瓜茎衰老过程中,西双版纳黄瓜茎在45天叶绿素含量最高,和叶片不同,西双版纳茎中叶绿素含量变化趋势不明显,茎叶绿素含量最高出现在45天,表明野生种茎可能还未进入衰老期。‘华黄5号’茎叶绿素含量在25天最高,茎中叶绿素含量先上升后下降；野生种茎中叶绿素含量高于栽培种。在栽培种和野生种茎里SOD活性变化均呈现双峰式变化趋势,西双版纳黄瓜和‘华黄5号’在35天时茎中的SOD活性最高。西双版纳黄瓜茎的POD活性一直呈上升趋势,‘华黄5号’的POD变化有呈现双峰趋势。在45天时,西双版纳黄瓜和‘华黄5号’的POD活性达到最大,并且西双版纳黄瓜茎中的POD活性始终高于‘华黄5号’。在35天时西双版纳和‘华黄5号’黄瓜茎中可溶性蛋白的含量最高,整体变化趋势是先升高后下降,在45天时最低。表明此时茎已经走向衰老。
3.脱镁叶绿素脱镁叶绿酸a水解酶(PPH)和脱镁叶绿酸a加氧酶酶(PAO)基因克隆
克隆的PPH和PAO基因碱基序列片段长度分别为265 bp和230 bp, BLAST核酸同源性在线分析表明,PPH片段与拟南芥(AT5G13800)、花椰菜(OL386R)、葡萄(XM002271131.1)和烟草(AM851012.1)的该家族基因分别具有87%、88%、80%和78%的核苷酸序列同源性,PAO片段与拟南芥(NM-114357.5)、葡萄(FJ799360.1)、花椰菜(AB470926)、毛果杨(XM002331446.1)和蓖麻(XM002523689)的该家族基因分别具有90%、86%、85%、83%和82%的核苷酸序列同源性,证明该扩增产物是PPH和PAO酶基因片段。
4.西双版纳和‘华黄5号’黄瓜叶衰老过程中PPH和PAO基因的相对表达量。
PPH和PAO基因在西双版纳黄瓜叶中的相对表达量的变化趋势有所不同,PPH基因的相对表达量是先上升后下降,在35天时表达量最高。PAO基因在15、25天时表达较弱,在35天时表达最强。PPH和PAO基因在‘华黄5号’叶的相对表达量与在西双版纳黄瓜叶片里表达模式基本相同,PPH基因的相对表达量是先上升后下降,在35天时表达量最高,PAO基因在15、25天时表达较弱,和在西双版纳不同的是,PAO基因在45天表达最强。这两个基因在西双版纳和‘华黄5号’叶片中表达呈正相关。
5.西双版纳和‘华黄5号’黄瓜茎衰老过程中PPH和PAO基因的相对表达量。
与在叶片中的表达模式相似,PPH和PAO基因在西双版纳黄瓜茎中的相对表达量的变化趋势也有所不同,PPH基因在茎中的表达趋势和在叶片中的表达趋势相同,先上升后下降,在35天时最强。PAO基因的表达变化趋势是先上升、后下降、再上升,在45天表达最强。和在西双版纳黄瓜茎里的表达模式不同,PPH和PAO基因在‘华黄5号’茎的表达模式是上升、稍微下降、上升、稍微下降。并且和5日的参照值相比都有一个较高的表达量。PPH在35天表达最强,PAO基因在15天表达最强。相关性分析表明,这两个基因在‘华黄5号’茎中表达呈正相关,而在西双版纳茎中表达呈负相关。
Chlorophyll is an important pigment in the plant chloroplasts being concerned with photosynthesis.Chlorophyll degradation is an important phenomenon in the senescence process. Chlorophyll is degraded in plants as a result of natural or induced senescence.Loss of green color is the most obvious sign of plant senescence.In this study, the cultivar Cucumis sativusL.var.Huahuang 5 and the wild species Cucumis sativus L.var.xishuangbannanesis Qi etYuan with different senescence were used as experient materials.The physiological and biochemical characteristics were preliminary studied during senescence in the wild species and the cultivar. Meanwhile, Two key enzymes gene of Chlorophyll degradation (Pheophytin Pheophorbide Hydrolase and Pheideaoxygenase) were cloned. Expression of these two genes during leaf and stem senescence was quantified through real-time PCR technique. The relationship between the expression model of PPH and PAO genes and anti-senescence were discussed. Major results are as follows:
1. The physiological and biochemical characteristics of cucumber leaf during senescence
As xishuangbannanesisQietYuan and Huahuang 5 became senescent, chlorophyll content in leaves increased first,but decreased later. The Chlorophyll content was the highest at 15d. The ageing symptoms of leaf in Huahuang 5 appeared at 35d, the contents at 45d were 45% at 15d. on the contrary, chlorophyll content in xishuangbannanesisQi etYuan was always a higer level. The activities of SOD in Huahuang 5 decreased dramatically first, but increased faint later, decreased at 35d again. The activities of SOD of xishuangbannanesisQi etYuan at 35d was the highest, decreased later, The activities of SOD in the wild species started to decrease 20 days later than the cultivar, Maybe the senescent of xishuangbannanesisQietYuan start at 35d.The activities of POD of Huahuang 5 all raise as cucumber became senescent, POD showed two peaks in the wild species, The activities of POD was higher than Huahuang 5 except at 5d. The activities of POD of Huahuang 5 and xishuangbannanesisQi etYuan was the highest at 45d. the ability of the wild species which cleaned out active oxygen was stonger than the cultivar at the later stage of cucumber growth. The soluble protein content of xishuangbannanesis Qi etYuan was higher than Huahuang 5, The Chlorophyll content was the highest at 35d. The soluble protein content of Huahuang 5 was at 45, meanwhile, the variation laws of the soluble protein was not significantly.
2.The physiological and biochemical characteristics of cucumber stem during senescence
As xishuangbannanesis QietYuan and Huahuang 5 became senescent, The Chlorophyll content in stem of xishuangbannanesisQi etYuan was the highest at 45d. unlike leaf, the variation trends of the Chlorophyll content in stem of xishuangbannanesis Qi etYuan was not significantly. Maybe the stem didn't enter the senescent.The Chlorophyll content in stem of Huahuang 5 was the highest at 25d. chlorophyll content in stems increased first, but decreased later.Thecontent of chl of xishuangbannanesisQi etYuan was higher than Huahuang 5.
The activities of SOD of xishuangbannanesisQi etYuan and Huahuang 5, showed two peaks. It were the highest at 35d.The activities of POD of xishuangbannanesis Qi etYuan all raise as cucumber became senescent, it showed two peaks in the cultivar, The activities of POD of Huahuan 5 and xishuangbannanesisQi etYuan was the highest at 45d. The activities of POD was higher than Huahuang 5.The soluble protein content of xishuangbannanesis Qi etYuan and Huahuang 5 were the highest at 35d, overall, the variation trends of the soluble protein was in stem increased first,but decreased later.the lowest were at 45d.
3. Cloning of PPH and PAO genes.
The length of cloned PPH and PAO genes were 265 base pairs and 230 base pairs, Analysis of the nucleotide sequenee by BLAST on line indicated that homology of PPH gene was up to 87% between Cucumis sativusL.var. and Arabidopsis thaliana (L.) Heynh, up to 88% between it and Brassica oleracea L. var. botrytis L, up to 80% between it and Vitis vinifera, up to 78% between it and Nicotiana tabacum. homology of PAO gene was up to 90% between Cucumis sativusL.var. and Arabidopsis thaliana (L.) Heynh, up to 86% between it and Brassica oleracea L. var. botrytis L, up to 85% between it and P. trichocarpa, up to 82% between it and Ricinus communis.
4. Relative quantification of PPH and PAO genes during leaf senescence of Cucumis sativus L.var.xishuangbannanesis Qi etYuan and Huahuang 5.
It is different that expression of both PPH and PAO genes in the leaf of xishuangbannanesisQi etYuan. The relative expression of PPH gene basically up-regulated first, down-regulated later. It was the highest expression at 35d. The minimum expression of PAO gene at 15d and 25d.The maximum at 35d. Expression of PPH and PAO genes between cultivars and wild species was quite similar. The relative expression of PPH gene basically up-regulated first,down-regulated later in stem of Huahuang 5. The maximum at 35d too, the minimum expression of PAO gene at 15d and 25d, the maximum at 45d. There was a positive correlation between both PPH and PAO genes in the leaf of xishuangbannanesis Qi etYuan and Huahuang 5.
5. Relative quantification of PPH and PAO genes during stem senescence of Cucumis sativus L.var.xishuangbannanesis Qi etYuan and Huahuang 5.
Unlike the expression modle of leaf. It is different that expression of both PPH and PAO genes in the stem of xishuangbannanesis Qi etYuan. Expression of PPH and PAO genes between leaf and stem was quite similar. The relative expression of PPH gene basically up-regulated first, down-regulated later too.it was the highest expression at 35d. The expression mold of PAO gene basically up-regulated first, down-regulated later, then up-regulated. The maximum at 45d. Unlike the expression modle of stem of xishuangbannanesis Qi etYuan, expression of both genes basically down-regulated first, up-regulated, down-regulated, up-regulated last, it showed two peaks in the cultivar,there was a higher expression than the value of at 5d. The maximum expression of PPH at 35d, PAO at 15d. There was a positive correlation between both PPH and PAO genes in the stem of Huahuang 5. negative correlation in the stem of xishuangbannanesis Qi etYuan.
引文
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