鹅掌楸LcPIN 1a基因的克隆及其对植株生长发育的影响OA北大核心CSTPCD
Cloning of the Liriodendron chinense LcPIN 1a genes and its effect on plant growth and development
[目的]PIN-FORMED(PIN)属于生长素转运蛋白,能够介导生长素的极性运输,在植物的生长和发育过程中扮演着关键角色.本研究旨在解析鹅掌楸PIN 1基因对植物生长和发育的影响.[方法]通过蛋白序列同源比对、系统发育树构建以及蛋白结构域预测的方法,鉴定鹅掌楸中PIN1 的同源蛋白LcPIN1s.然后,通过转录组数据解析LcPIN 1s基因的组织特异性表达情况,并通过实时荧光定量逆转录PCR(RT-qPCR)方法研究LcPIN 1s在不同苗龄再生植株的根、茎和叶组织中的表达动态.此外,通过转录组数据解析LcPIN 1s基因在低温、高温以及干旱胁迫条件下的时序动态表达,并进一步利用RT-qPCR方法研究LcPIN 1s响应干旱胁迫与内源ABA合成之间的关联.最后,通过克隆鹅掌楸叶片中高表达的PIN 1同源基因LcPIN 1a,构建由CaMV35S启动子驱动的过表达载体(35S:LcPIN 1a),通过异源转化拟南芥(Arabidopsis thaliana)和同源转化杂种鹅掌楸(Liriodendron×sinoamericanum),筛选并获得的异源过表达(LcPIN 1a-HO)和同源过表达(LcPIN 1a-OE)阳性再生植株,分别进行生长和发育性状的测定和分析.[结果]通过生物信息学方法在鹅掌楸基因组中成功鉴定到了3 个PIN1 同源蛋白,分别命名为LcPIN1a、LcPIN1b和 LcPIN1c.组织特异性表达分析显示,LcPIN1a主要在叶片中高表达,而LcPIN 1b和LcPIN 1c主要在茎和根以及成年后的雌蕊中高表达.另外,鹅掌楸 3 个PIN 1同源基因均受到低温(4℃)和干旱(质量分数 15%PEG6000)处理的诱导而表现出先上调后下调的表达模式,但在高温(40℃)胁迫下则会急剧下调表达.利用聚乙二醇(PEG)、脱落酸(ABA)以及 ABA 合成抑制剂氟啶酮(Flu)处理,发现LcPIN 1s在响应干旱诱导时表现出不同的模式,即LcPIN 1a不依赖于内源ABA的合成,而LcPIN 1b和LcPIN 1c则依赖于内源ABA的合成.最后,通过对拟南芥异源过表达株系(LcPIN 1a-HO)再生植株的生长性状统计分析发现,其在根长和株高方面均显著低于野生型,同时雄蕊数发生显著变异,从野生型的 6 枚雄蕊变为以 5 枚为主.在杂交鹅掌楸过表达株系(LcPIN 1a-OE)中,其体胚成苗率显著降低,并且成苗后的再生植株在根长和株高方面均显著低于野生型,同时根系结构发生明显变化,主根不明显.[结论]鹅掌楸PIN1 蛋白在植株的营养和生殖生长方面发挥重要作用,过量表达不利于植株的正常生长和发育.
[Objective]This study aimed to explore the role of the auxin transporter PIN1 in plant growth and development in Liriodendron chinense.[Method]Three PIN1 homologous proteins were identified in the Liriodendron genome using bioinformatic methods,and expression pattern analyses of the three LcPIN 1s genes were performed in different tissues and response to various abiotic stresses.An overexpression vector driven by the CaMV35S promoter was then constructed and transformed into Arabidopsis and Liriodendron×sinoamericanum,followed by phenotypic determination of growth and developmental traits in the transgenic positive lines.[Result]Three PIN1 homologous proteins were identified in the Liriodendron genome,named LcPIN1a,LcPIN1b and LcPIN1c.Expression pattern analyses showed that LcPIN 1a was mainly expressed in leaves,while LcPIN 1b and LcPIN 1c were primarily expressed in roots and stems and stigmas when the plantlets transitioned into reproductive growth.In addition,all three LcPIN 1genes transcriptionally responded to drought stress,with LcPIN 1b and LcPIN 1c showing dependence on the biosynthesis of endogenous ABA,while LcPIN 1a does not.Root length and plant height were significantly reduced in LcPIN 1a-heterologous overexpression(LcPIN 1a-HO)lines compared to wild-type Arabidopsis.The number of stamens was predominantly five in LcPIN 1a-HO lines,whereas wild-type Arabidopsis typically contained six stamens.The regeneration of plantlets in LcPIN 1a-overexpressing(LcPIN 1a-OE)Liriodendron×sinoamericanum was significantly reduced compared to wild-type plants.In addition,the root length and plant height of LcPIN 1a-OE regenerated seedlings were significantly lower than those of the wild type.The root structure of LcPIN 1a-OE plants was significantly changed,with the taproot being less distinct.[Conclusion]The PIN1 proteins of L.chinense play a crucial role in vegetative and reproductive growth.Overexpression of LcPIN 1genes can be detrimental to normal plant growth and development.
郝兆东;马筱筱;王丹丹;陆叶;施季森;陈金慧
林木遗传育种全国重点实验室,南方现代林业协同创新中心,南京林业大学林草学院,江苏 南京 210037
林学
鹅掌楸生长素转运蛋白PIN1基因生长发育
Liriodendron chinenseauxin transporterPIN 1genegrowth and development
《南京林业大学学报（自然科学版）》 2024 (006)
51-61 / 11
国家自然科学基金面上项目(32071784).
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