CN117343940A - 一种杂交鹅掌楸NAC转录因子LhSND1及其应用 - Google Patents
一种杂交鹅掌楸NAC转录因子LhSND1及其应用 Download PDFInfo
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Abstract
本发明公开了一种杂交鹅掌楸NAC转录因子LhSND1及其应用，属于植物基因工程技术领域。本发明所克隆的NAC转录因子基因LhSND1，其核苷酸序列如SEQ ID NO.1所示，编码的氨基酸序列如SEQ ID NO.2所示。本发明通过多序列比对、系统进化分析与表达模式研究，揭示了LhSND1基因的序列特征及生物学功能。本发明还提供了所述NAC转录因子基因在促进次生壁生物合成的应用手段。本发明所克隆的LhSND1基因将在杂交鹅掌楸木材材性遗传改良中发挥重要的作用。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一种杂交鹅掌楸NAC转录因子LhSND1基因及其在促进次生壁形成中的应用。
背景技术
转录因子是一类能与真核生物基因启动子上的顺式作用元件发生特异性结合从而调控基因表达的蛋白质。植物次生壁含有丰富的木质素、纤维素和半纤维素，构成木材生物质的主体部分。植物体中存在复杂的多层级转录调控网络作用于次生壁的合成，现有研究表明，众多转录因子在次生壁合成过程中发挥中重要的功能。
NAC(NAM，ATAF1/2和CUC2)转录因子是植物特有的一类含有NAM结构域的转录调控因子。在拟南芥中，NAC家族成员NST1、NST2和SND1/NST3是较早发现的调控植物次生壁生物合成的关键因子。其中，SND1位于次生壁合成调控网络的上游，与下游转录因子MYB46、MYB103和KNAT7等协同调控木质素、纤维素和半纤维素合成基因的表达。杨树NAC类转录因子PtrWNDs是SND1的同源蛋白，过表达PtrWNDs能够激活次生壁相关转录因子和合酶基因的表达，导致次生细胞壁的异位沉积。
杂交鹅掌楸(Liriodendron hybrids)是以鹅掌楸属两个仅存种，即中国鹅掌楸(Liriodendron Chinense(Hemsl.)Sarg.)和北美鹅掌楸(Liriodendron tulipifera L.)为亲本所创造的种间杂种。与两个亲本种相比，杂交鹅掌楸生长速度快、干形饱满通直，具有很强的生长优势，是园林绿化和用材林营建的重要树种。目前对于杂交鹅掌楸NAC转录因子的研究相对缺乏，开展关于杂交鹅掌楸NAC转录因子调控次生壁形成的分子机制研究，将为杂交鹅掌楸材性改良奠定基础。
发明内容
针对现有技术存在的上述问题，本发明的目的是提供一种杂交鹅掌楸NAC转录因子LhSND1基因。本发明的另一目的是提供杂交鹅掌楸转录因子LhSND1基因在促进次生壁形成中的应用。
为了实现上述发明目的，本发明采用下述技术方案：
一种杂交鹅掌楸NAC转录因子LhSND1基因，其核苷酸序列如SEQ ID NO.1所示。
杂交鹅掌楸转录因子LhSND1基因的表达蛋白，其氨基酸序列如SEQ ID NO.2所示。
本发明提供了一种含有所述杂交鹅掌楸NAC转录因子LhSND1基因的过表达载体。
本发明还提供了一种含有杂交鹅掌楸NAC转录因子LhSND1基因的过表达载体的构建方法，将如SEQ ID NO.1所示的LhSND1基因的全长编码区序列克隆到pBI121载体中CaMV35S启动子的下游，构建过表达植物载体pBI121-LhSND1。
本发明还提供了一种在nst1/snd1双突变体拟南芥中表达LhSND1基因的方法，将过表达载体pBI121-LhSND1，通过浸花法遗传转化nst1/snd1双突变体拟南芥植株。
本发明还提供了杂交鹅掌楸NAC转录因子LhSND1基因在促进次生壁形成中的应用，更具体的是在激活次生壁生物合成基因中的应用。
本发明的有益效果：与现有技术相比，本发明从杂交鹅掌楸中克隆得到一条NAC转录因子基因LhSND1，通过遗传转化使LhSND1基因在拟南芥nst1/snd1双突变体植株中表达，对转基因拟南芥植株的表型差异和组织切片染色的研究发现，LhSND1基因的表达能够恢复nst1/snd1双突变体拟南芥严重倒伏、无法直立生长的缺陷，并使次生壁的合成也恢复正常，可见LhSND1基因具有促进次生壁形成的功能，在林木材性的遗传改良中将具有重要的应用价值。
附图说明
图1是杂交鹅掌楸LhSND1基因PCR扩增的凝胶电泳图。
图2是杂交鹅掌楸LhSND1蛋白保守结构域的预测图。
图3是杂交鹅掌楸LhSND1基因的系统进化分析图。
图4是杂交鹅掌楸LhSND1基因的表达模式图。
图5是拟南芥植株的生长表型图。
图6是拟南芥植株茎的横切图。
图7是拟南芥次生壁合成基因的表达模式图。
具体实施方式
下面结合具体实施例对本发明进一步详细说明。
实施例1杂交鹅掌楸LhSND1基因的克隆以及LhSND1过表达载体的构建
1、杂交鹅掌楸LhSND1基因的克隆
在杂交鹅掌楸转录组数据库中比对到一条NAC转录因子家族的同源基因，使用ORFFinder(http://www.ncbi.nlm.nih.gov/gorf.Html)工具发现该基因全长包含完整的编码区。设计LhSND1基因完整编码区的上下游引物LhSND1-F(ATGGATGACATGAGTCTGTCGGTG)和LhSND1-R(TCATACGTGGTCCGATGTTGATGATG)，以杂交鹅掌楸cDNA为模板进行PCR扩增，PCR产物经1％琼脂糖凝胶电泳检测得到一条长约1200bp的条带，如图1所示。PCR产物经纯化回收后，送生工公司测序验证，最终获得LhSND1基因编码区全长序列如SEQ ID NO.1所示，其编码蛋白的氨基酸序列如SEQ ID NO.2所示。
2、LhSND1过表达载体的构建
在LhSND1基因上下游分别引入pBI121载体上Sma I酶切位点两侧的同源互补序列，作为PCR引物(pBI-LhSND1-F：CTCTAGAGGATCCCCATGGATGACATGAGTCTGTCGGTG，pBI-LhSND1-R：AGGGACTGACCACCCTACGTGGTCCGATGTTGATG)，以杂交鹅掌楸cDNA为模板，PCR扩增带有pBI121载体同源互补序列的LhSND1基因片段。进而利用infusion同源融合法将LhSND1基因全长克隆到pBI121载体中CaMV 35S启动子的下游，构建过表达植物载体pBI121-LhSND1。通过电击法将LhSND1过表达载体转入EHA105农杆菌中，经PCR检测和测序验证后保存菌种备用。
实施例2杂交鹅掌楸LhSND1基因的序列特征与进化树分析
1、LhSND1基因序列的基本特征分析
应用ProtParam(http://www.expasy.org/tools/protparam.htmL)程序对LhSND1蛋白的理化性质进行在线分析发现，LhSND1基因全长包含1209bp的开放阅读框(ORF)，编码由402个氨基酸残基组成的多肽，等电点(PI)为6.24，相对分子量(MW)为45.56kD。应用NCBI网站的CDD(Conserved Domain Database)数据库，对LhSND1蛋白可能存在的功能结构域进行预测，发现LhSND1蛋白在17-143aa处存在一个NAM结构域，表明LhSND1属于NAC转录因子家族。
2、LhSND1与拟南芥NAC家族基因的系统发育进化分析
应用MEGA7软件对LhSND1与10个拟南芥NAC基因(VND1-AT2G18060、VND2-AT4G36160、VND3-AT5G66300、VND4-AT1G12260、VND5-AT1G62700、VND6-AT5G62380、VND7-AT1G71930、NST1-AT2G46770、NST2-AT3G61910、SND1/NST3-AT1G32770)构建系统发育进化树(图3)，结果显示LhSND1基因与拟南芥NST亚家族基因位于同一分支上，与其中的拟南芥SND1/NST3基因同源性最高，推测LhSND1基因可能与次生壁形成相关。
实施例3杂交鹅掌楸LhSND1基因的功能研究
1、LhSND1基因的表达模式分析
为了研究LhSND1基因在杂交鹅掌楸不同组织中的表达模式，收集6个月大杂交鹅掌楸土培苗的根(RT)、顶芽(AB)、第1茎节的茎(IN1)、第3茎节的茎(IN3)、第5茎节的茎(IN5)、第1至第3茎节处的叶片(YL)、第5茎节处的叶片(ML)，分别提取这些组织的RNA，并反转录成cDNA，用于实时荧光定量PCR分析。结果如图4所示，LhSND1基因在杂交鹅掌楸根、顶芽、茎和叶片中都有不同程度的表达，在第1茎节、第3茎节、至第5茎节中的表达量持续上升，在成熟茎中的表达量最高，且在成熟叶中的表达要高于在幼叶中的表达。这些结果表明LhSND1基因具有一定的组织表达特异性，可能参与杂交鹅掌楸生长发育过程。
2、在nst1/snd1双突变体拟南芥中表达LhSND1基因
将pBI121-LhSND1农杆菌活化至菌液OD600达到0.4-0.5，离心收集菌体并重悬于1/2MS液体培养基中，用于拟南芥侵染。选择生长旺盛、处于初花期的nst1/snd1双突变体拟南芥作为待转化植株，利用浸花法进行遗传转化。最后，通过分子检测和抗性筛选获得T3代转基因拟南芥。对nst1/snd1双突变体拟南芥(nst1/snd1)、野生型拟南芥(WT)与转LhSND1基因的回补突变体拟南芥(nst1/snd1+LhSND1)植株的表型进行观察，如图5所示，nst1/snd1双突变体拟南芥植株出现严重倒伏、无法直立生长，而转LhSND1基因的回补突变体拟南芥恢复直立生长，生长状态与野生型拟南芥基本一致。
3、不同拟南芥植株茎次生壁的形成情况
为了研究不同拟南芥植株的维管组织木质部(xy,xylem)和束间纤维(if,interfascicular)发育情况，将nst1/snd1双突变体(nst1/snd1)、转LhSND1基因的回补突变体(nst1/snd1+LhSND1)及野生型拟南芥(WT)同时种植，生长7-8周后，分别取基部茎横切，并进行HCl-间苯三酚染色。如图6所示，与野生型拟南芥相比，nst1/snd1双突变体茎中维管束间细胞(if)没有观察到木质素染色，说明其没有次生壁结构，而转LhSND1基因的回补突变体茎中维管束间细胞(if)发育正常，次生壁形成缺陷的表型完全恢复。
4、次生壁合成基因的表达量分析
提取6个月大野生型、nst1/snd1双突变体及转LhSND1基因的回补突变体拟南芥的RNA，反转录为cDNA，对拟南芥次生壁生物合成相关基因的表达量进行荧光定量PCR分析。结果如图7，以每个基因在野生型拟南芥中的表达量作为参照，木质素合成的酶类基因CCR1、4CL1、CCoAOMT，纤维素合酶基因CesA4、CesA7、CesA8，以及半纤维素生物合成相关基因FRA8、IRX8、IRX9的表达量在nst1/snd1双突变体中均显著下调，但在转LhSND1基因的回补突变体中的表达量与野生型拟南芥没有显著差异。以上结果表明LhSND1基因的表达能够激活拟南芥的次生壁生物合成基因的表达。
本发明公布的杂交鹅掌楸LhSND1基因开放阅读框全长1209bp，编码由402个氨基酸残基组成的多肽，在其N端第17-143个氨基酸上存在一个高度保守的NAM结构域。LhSND1基因与拟南芥SND1/NST3基因有很高的同源性。在nst1/snd1双突变体拟南芥中表达LhSND1基因，能够激活拟南芥次生壁生物合成基因的表达，进而恢复突变体维管束间细胞次生壁形成的缺陷，使拟南芥植株恢复直立生长，可见LhSND1基因具有促进次生壁合成的功能，可用于林木材性的遗传改良。
Claims (6)
1.一种杂交鹅掌楸转录因子LhSND1基因，其特征在于，核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的杂交鹅掌楸转录因子LhSND1基因的表达蛋白，其特征在于，氨基酸序列如SEQ ID NO.2所示。
3.一种含有权利要求1所述杂交鹅掌楸转录因子LhSND1基因的表达载体。
4.一种含有杂交鹅掌楸转录因子LhSND1基因的表达载体的构建方法，其特征在于，将如SEQ ID NO.1所示的LhSND1基因的全长编码区序列克隆到pBI121载体中CaMV 35S启动子的下游，构建过表达植物载体pBI121-LhSND1。
5.权利要求1所述的杂交鹅掌楸转录因子LhSND1基因在促进植物次生壁形成中的应用。
6.权利要求1所述的杂交鹅掌楸转录因子LhSND1基因在激活次生壁合成基因中的应用。
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