枳砧红绵蜜柚嫁接黄化苗的光合特性及其CgSGR基因克隆与表达分析

Photosynthetic Characteristics of Etiolated Seedling of Citrus grandis(L.)Osbeck. cv. ‘Hongmian miyou' Grafted to Poncirus trifoliata and Expression of CgSGR

该研究以枳砧‘红绵蜜柚’(Hm/Pt)为试验材料,以枳砧‘琯溪蜜柚’(Gx/Pt)和香橙砧‘红绵蜜柚’(Hm/Cj)为对照,测定分析3种砧穗组合蜜柚的光合特性、叶绿素含量差异;利用RT-PCR技术克隆并分析滞绿基因(CgSGR)表达,为进一步研究枳砧‘红绵蜜柚’嫁接不亲和机理奠定基础。结果显示:(1)枳砧‘红绵蜜柚’嫁接苗净光合速率、气孔导度、蒸腾速率、叶肉羧化效率明显低于2个对照组合,且3种砧穗组合以上各指标的最大值均出现在嫁接后第98天。(2)3个嫁接组合的叶绿素含量变化趋势一致,但枳砧‘红绵蜜柚’在整个试验期间的叶绿素a、叶绿素b和总叶绿素显著低于2个对照组合。(3)成功克隆获得‘红绵蜜柚’滞绿蛋白SGR调控基因序列,命名为CgSGR(GenBank登录号:MF945620);CgSGR基因片段长度为816bp,序列编码一个含有271个氨基酸的蛋白质,分子量为30.62kD,属于稳定的碱性亲水性蛋白;SGR序列多重比对结果显示,‘红绵蜜柚’CgSGR序列与柑橘属柚、甜橙和克里曼丁橘之间一致性较高,其中‘红绵蜜柚’与柚无碱基差异,与甜橙在第33bp处有1个变异位点,与克里曼丁橘在33、52、118、159、622和765bp处有6个变异位点;系统进化树表明,CgSGR与柑橘属甜橙、克里曼丁橘的距离最近,与荔枝关系较近,与其它物种的关系较远。(4)实时荧光定量PCR结果表明,CgSGR基因在香橙砧‘红绵蜜柚’中的表达量总体均低于其他2个组合材料,而嫁接139d后,3个材料均随着植株黄化,CgSGR基因的表达水平均呈上升趋势,且枳砧‘红绵蜜柚’的CgSGR基因表达显著高于2个对照组合。(5)随着嫁接苗的生长发育,CgSGR的表达水平总体变化趋势与净光合速率、叶绿素的变化趋势相反。研究认为,枳砧‘红绵蜜柚’CgSGR基因的表达量在嫁接苗叶绿素降解过程中上调,其可能参与调控黄化幼苗的叶绿素降解。

In order to discuss the etiolation mechanism and provide a theoretical basis in citrus breeding programs,we studied the relationship among photosynthetic characteristics,photosynthetic pigment content and the relative level of CgSGRexpression of three graft combinations.Citrus grandis（L.）Osbeck.cv.‘Hongmian miyou＇（Hm/Pt）and‘Guanxi miyou＇grafted on Poncirus trifoliata（Gx/Pt）,and‘Hongmi-an miyou＇grafted on C.junos（Hm/Cj）were used as the experimental materials.The results were showed as follows.（1）The photosynthetic rate,stomatal conductance,transpiration rate and mesophyll carboxylation efficiency of Hm/Pt were significantly lower than that of Gx/Pt and Hm/Cj;（2）The three graft combinations had similar change trend of chlorophyll content,while the chlorophyll a,b and total chlorophyll in Hm/Pt were significantly lower than that of control groups;（3）The open reading frame（ORF）of CgSGR was 816 bp,encoding 271 amino acids（GenBank accession number：MF945620）.The deduced protein molecular weight was 30.62 kD,which was a stable alkaline hydrophilic protein.According to multiple sequence alignment analysis,the sequence of‘Hongmian miyou＇and‘Guanxi miyou＇were high consistency with C.grandis,C.sinensis and C.clementina.Based on phylogenetic tree analysis,CgSGR keep closest genetic relationship with C.grandis,C.sinensis and C.clementina,and relatively close with Litchi chinensis;（4）RT-qPCR results showed that CgSGRexpression levels were up-regulated in Hm/Pt when the plants initiated etiolation.The CgSGRtranscripts reached the highest level grafted after 404 days;（5）With the growth of the grafted seedlings,the trend of expression level of CgSGR was opposite with the net photosynthetic rate and chlorophyll content.The results indicated that CgSGR might be involved in transcriptional regulation during the seedling etiolation of C.grandis ‘Hongmian miyou＇grafted to P.trifoliata.