CN117305356A - 84K杨PagAPA1基因在树木次生木质部发育中的调控作用 - Google Patents
84K杨PagAPA1基因在树木次生木质部发育中的调控作用 Download PDFInfo
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Abstract
84K杨PagAPA1基因在树木次生木质部发育中的调控作用，属于基因工程技术领域。本发明为了解决木材数量及品质的需要日渐增大，84K杨PagAPA1生物功能未知的问题，通过农杆菌介导的84K杨遗传转化系统获得了PagAPA1过量表达84K杨转基因植株，再利用一系列技术手段分析发现，PagAPA1的过量表达影响了植株的生长发育及木材的形成，主要体现在该基因影响树木次生细胞壁的木质素的沉积，进而改变木质部细胞的形态结构及分布，以上结果说明PagAPA1对树木次生木质部的发育具有一定调控作用。本发明研究内容对培育优质木材林木新品种具有重要的理论指导意义。
Description
技术领域
本发明属于基因工程技术领域，具体涉及84K杨PagAPA1基因在树木次生木质部发育中的调控作用。
背景技术
树木的次生生长对人类的生产和生活具有重大意义。次生木质部，即木材作为全球最为丰富的可再生生物质能源，在建筑业、制浆造纸以及纺织业等方面具有广泛的应用和重要的经济价值。
植物的次生生长及木材形成十分复杂，是由维管形成层启动的一系列生物学过程，包括维管组织细胞的持续分裂与分化、次生细胞壁形成、木质化以及细胞程序化死亡和心材形成等过程，是一个不可逆的且受高度时空调控的次生发育过程。木材的主要由加厚的次生细胞壁发生，这些次生细胞壁在木质部细胞膨胀停止后开始沉积，主要由纤维素、半纤维素和木质素组成。这一过程受多种碳水化合物活性酶的协同表达来驱动，同时由多层转录因子调控网络控制，还受到各种遗传和环境信号的动态调控。已鉴定多种与次生细胞壁沉积相关的调控因子，包括半胱氨酸蛋白酶、类受体激酶和渗透素-like蛋白等。尽管新的证据揭示了参与次生细胞壁沉积调控的各种信号，但全面的通路尚未完全建立，是否还存在其他信号通路还有待探索。
APA1天冬氨酸蛋白酶A1(spartic proteinase A1)是一类重要的蛋白水解酶，其在植物生长发育、抗病及逆境胁迫过程中发挥着重要的信号调控作用。木本植物茎中已发现该基因，但其生物功能迄今为止从未被研究。
发明内容
本发明提供了84K杨PagAPA1基因在调控树木次生木质部发育中的应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种84K杨PagAPA1基因，所述PagAPA1基因的核苷酸序列如SEQ IDNo.1所示。
本发明还提供了一种所述基因编码的蛋白APA1，所述蛋白质的氨基酸序列如SEQID NO.2所示。
本发明还提供了一种表达载体，包括所述的PagAPA1基因。
本发明还提供了一种重组菌，包括所述的PagAPA1基因或所述的表达载体。
本发明还提供了一种所述的PagAPA1基因、所述的基因编码蛋白、所述的表达载体或所述的宿主在调控树木次生木质部发育中的应用。
优选的，所述调控树木次生木质部发育的方法为在84K杨中过表达所述PagAPA1基因。
优选的，所述调控次生木质部发育为转基因84K杨生长迟缓，茎干木质部宽度增加，茎干木质部面积占茎面积的比率增加，木质素合成增加。
本发明首次发现证明了PagAPA1基因在调控树木次生木质部发育中具有重要作用，为进一步理解木材形成的分子调控机制，利用分子育种技术进行林木材性遗传改良提供了思路。
本发明具有以下有益效果：
本发明通过农杆菌介导的84K杨遗传转化系统获得了PagAPA1过量表达84K杨转基因植株。利用一系列技术手段分析发现，PagAPA1的过量表达使84K杨生长迟缓，茎干木质部宽度增加，茎干木质部面积占茎面积的比率增加，木质素合成增加。进而发现PagAPA1基因对树木次生木质部的发育具有一定调控作用，主要体现在该基因通过影响树木次生细胞壁的木质素的沉积，进而改变木质部细胞的数量和分布，影响植株的生长发育及木材的形成(见附图1至附图6)。本发明研究内容对培育优质木材林木新品种具有重要的理论指导意义。
附图说明
图1为本发明以18SrRNA基因为内参基因，对比野生型84K杨和APA1过表达株系中的相对表达量；图中OE1、OE2分别表示过表达植株2个不同株系，CK代表野生型84K杨，误差线代表由至少三个生物学重复计算的标准误，星号代表t检验结果，*P<0.05，***P<0.001。
图2为本发明PagAPA1过量表达及野生型植株温室生长2个月后的生长状况图；图中OE1、OE2分别表示过表达植株2个不同株系，CK代表野生型84K杨，比例尺为10cm；
图3为本发明PagAPA1过量表达植株温室生长4个月后取第16茎节进行硬组织切片的结果图；图中OE1、OE2分别表示过表达植株2个不同株系，CK代表野生型84K杨；使用番红固绿染色，可以观察植株不同组织形态，染色后，组织木质化、木栓化的细胞壁染成红色，纤维素细胞壁染成绿色，导管染成红色，筛管染成绿色；放大倍数为40倍；
图4为本发明温室生长4个月的转基因植株木质部的宽度结果图；图中OE1、OE2分别表示过表达植株2个不同株系，CK代表野生型84K杨；误差线代表由至少三个生物学重复计算的标准误，星号代表t检验结果，**P<0.01；
图5为本发明温室生长4个月的转基因植株木质部占整个茎面积的比率分析结果图；图中OE1、OE2分别表示过表达植株2个不同株系，CK代表野生型84K杨；误差线代表由至少三个生物学重复计算的标准误，星号代表t检验结果，**P<0.01；
图6为本发明温室生长4个月的转基因植株次生细胞壁成分分析结果图；图中OE1、OE2分别表示过表达植株2个不同株系，CK代表野生型84K杨；误差线代表由至少三个生物学重复计算的标准误，星号代表t检验结果，*P<0.05，***P<0.001。
具体实施方式
下面通过具体实施方式对本发明进行详细说明，本发明的特点和优点将随着这些说明而变得更为清楚、明确。
下面结合具体实施例对本发明作进一步的详细描述，以便本领域技术人员理解。以下实施例中如无特别说明均为常规方法。所使用的材料、试剂、酶、感受态细胞、质粒等，如无特殊说明，均可从商业途径得到。
实施例
实施例1：84K杨PagAPA1基因的获得及植物表达载体的构建
1.PagAPA1基因序列的克隆
利用植物RNA提取试剂盒(诺唯赞)提取84K杨(Populus alba×Populusglandulosa)野生型植株总RNA；利用反转录试剂盒(Promega)进行反转录获得cDNA；以cDNA为模板，使用高保真酶(诺唯赞)与两端引物进行PCR扩增，反应完成后，电泳检测并使用琼脂糖凝胶回收试剂盒(天根)回收目的片段。
参照phytozome网站提供的84K杨基因组序列信息，在目的基因序列两端设计基因特异性引物并委托生物公司合成引物，在引物序列中加入XbaI酶切位点以及载体上的重组同源片段，获得用于PagAPA1基因克隆的上、下游引物序列如SEQ ID No.3和SEQ ID No.4所示。
SEQ ID No.3:
84K杨PagAPA1-F:
agaacacgggggactATGGGAGTGAACTTGAAAGCAATTG
SEQ ID No.4:
84K杨PagAPA1-R:
acccccggggatcctAGCTGCCTCAGCAAATCCAACT
2.植物表达载体的构建
本实验使用PBI121载体，使用XbaI限制性内切酶酶切，并回收载体片段，与PCR回收产物进行连接，转入DH5α大肠杆菌感受态细胞内，于LB固体抗性培养基(含50mg/L卡那霉素)上进行筛选，构建含有APA1全长CDS的过表达载体。挑取阳性菌落，扩大培养后提取质粒，验证序列是否正确。
实施例2：84K杨PagAPA1过表达植株的获得
1.转化农杆菌GV3101、侵染愈伤组织
将提取的质粒转化农杆菌GV3101菌株，在抗性LB固体培养基平板(含50mg/L卡那霉素，50mg/L利福平)上进行筛选。挑取阳性菌株扩大培养，将扩大培养的农杆菌菌株分装至50mL无菌离心管中，于离心机中以4℃，5000rpm的转速离心10min，弃去上清液，保留菌体，向离心管中加入50mL重悬液备用。取84K杨愈伤组织用于侵染，获得过表达株系。
2.转基因植株的鉴定
将转基因植株和生长状态一致的野生型植株种入土中，培养至4个月大，利用植物RNA提取试剂盒(诺唯赞)提取转基因植株和野生型植株总RNA，利用反转录试剂盒(Promega)进行反转录获得cDNA，使用荧光定量PCR试剂盒(Takara TBPremix ExTaqTMRR420A)和QuantStudio 6Flex Real-Time PCR System对cDNA进行实时荧光定量PCR检测，上游引物和下游引物核苷酸序列分别为SEQ ID No.5和SEQ ID No.6。内参基因为18SrRNA，步骤参照仪器的操作手册以及试剂盒的使用说明。对比野生型84K杨和APA1过表达84K杨中的相对表达量，具体如图1所示。
PCR引物序列如下：
SEQ ID No.5:
qPCR-APA1-F:AGTCATCTGGTGTTCTTGGTG
SEQ ID No.6:
qPCR-APA1-R:TCTTGTGTCTGGTTCTGCTTC
通过农杆菌介导的遗传转化的方式共获得了2个独立抗性株系，分别命名为APA1-OE1和APA1-OE2，过量表达及野生型植株温室生长2个月后的生长状况图如图2所示。
经过转录水平鉴定后，与84K杨野生型植株相比，PagAPA1基因在两株抗性植株中转录水平均有明显上升，表明成功获得PagAPA1基因的过量表达转基因84K杨植株。
实施例3：84K杨PagAPA1基因在树木次生木质部发育中的调控作用(表型分析)
1.木材硬组织切片的观察
分别取温室生长4个月的野生型84K杨及PagAPA1过表达植株茎干的第16茎节进行石蜡切片及组织化学染色分析，采用番红固绿染色，染色后组织木质化、木栓化的细胞壁染成红色，纤维素细胞壁染成绿色；导管染成红色，筛管染成绿色。利用电子显微镜(放大40倍)观察植物组织的不同组织形态(图3)。结果表明，过表达PagAPA1转基因84K杨植株的木质部发育超过对照，次生细胞壁比野生型厚，表明PagAPA1的过表达影响了植株次生生长过程。
2.茎横切面面积的比较
根据ImageJ软件测定木质部面积，再除以总面积得到木质部与茎总面积的比值，如图4可以看出过表达PagAPA1转基因84K杨植株的木质部面积与茎面积的比值比野生型植株的更高。同时，过表达PagAPA1转基因84K杨植株的木质部宽度较野生型植株更大(图5)。
3.次生细胞壁成分的分析
将植株茎干烘干、粉碎，加入72％浓硫酸30℃水浴锅中水解60min，加H2O使硫酸稀释至4％，充分混匀。加入SRS溶液(葡萄糖：3g/L，木糖：0.8g/L，阿拉伯糖：0.4g/L)，将含有样品和SRS的试管放入灭菌锅中，121℃灭菌1h，冷却至室温后使用真空泵过滤得水解液和残渣。
用热蒸馏水洗涤残渣至洗液呈中性后，105℃烘干、称重，在550℃下灰化，得灰分与漏斗总重，计算木质素含量。采用HPLC法分析水解液，根据色谱结果计算纤维素、半纤维素含量。从图6中可以看出，PagAPA1过表达84K杨的木质素含量较野生型的高，纤维素含量较野生型相当，而半纤维素含量较野生型的低。
综合以上所有结果，PagAPA1基因参与杨树次生木质部发育的调控过程。该基因可以促进木质素的合成，增强木质素的沉积，增强了茎干导管细胞的发育，影响植株茎干细胞的形态结构及分布，进而影响植株发育。
以上结合具体实施方式和/或范例性实例以及附图对本发明进行了详细说明，不过这些说明并不能理解为对本发明的限制。本领域技术人员理解，在不偏离本发明精神和范围的情况下，可以对本发明技术方案及其实施方式进行多种等价替换、修饰或改进，这些均落入本发明的范围内。本发明的保护范围以所附权利要求为准。
SEQ ID NO.1
ATGGGAGTGAACTTGAAAGCAATTGTGGGGTTTGTGTTTCTTTCATTTCTGTTGTTTGCTGTTGTGTCATCCGCGTCGAATGATGGTTTGCTTAGAATAGGATTAAAAAAGGTGAAACTTGATAAGAACAACCGAATTGCTGCACGGCTTGATTCCAAAGAGACCTTGAGAGCTTCTATTAGGAAATATAATCTTTGCAGTAATCTTGGAGAATCCGGGGATACTGATATTGTTGCATTGAAGAATTATCTGGATTCTCAGTACTATGGCGAGATTGGTGTTGGCTCTCCTCCGCAGAAGTTCACTGTGATCTTTGACACTGGTAGCTCCAATTTGTGGGTGCCATCATCTAAGTGCTATTTATCGGTTGCTTGTTACTTCCATTCCAAGTATGACTCTGGAAAATCAAGTACCTACAAGAAGAATGGAAAAAGTGCTGAAATTAGCTATGGCAGTGGATCTATTTCTGGTTTCTTCAGCAATGACGCTATTGAAGTTGGTGGCCTGGTTGTGAAAGATCAGGAATTTATTGAGGCAACTAAGGAGCCTAGTATCACATTTTTGGCGGCCAAGTTTGATGGTATATTGGGACTTGGGTTCAAAGAAATTTCAGTGGGAGATGCTGTGCCTGTCTGGGACAACATGATTAAACATGGTCTTATCAAGGACCCAGTATTTTCTTTCTGGCTTAACCGCAATGCAGAGGATGAAGAAGGGGGCGAAATTGTATTTGGTGGGATGGACCCGAAACATTACAAGGGCAAGCACACTTTTGTTCCTGTGACACAGAAAGGCTATTGGCAGTTCAACATGGGTGATGTCCATATTGGTGATAAACCAACTGGGTATTGTGCCAGCGGTTGTGCTGCAATTGCAGATTCTGGAACTTCCTTGTTGGCAGGTCCAACGACTATAATTACCATGATAAACCAAGCCATTGGAGCCTCTGGAGTTGTTAGCCAGCAATGCAAGGCTGTTGTTTCACAATATGGGGAAGCCATAATGGATTTGCTACTCTCTGAGGCACAGCCAAAGAGGATTTGCTCTCAAATTGGACTTTGCACATTTGATGGAACCCGTGGCATAAGCATTAGCATTCAGAGCGTGGTAGATGAGGGCAATGACAAGTCATCTGGTGTTCTTGGTGATGCTATGTGCTCTGCTTGTGAAATGGCAGTTGTCTGGATGCGAAGCCAGCTGAAGCAGAACCAGACACAAGATCGTATTTTGGACTATGCGAGTCAGCTTTGTGAAAGAATGCCAAACCCAATGGGAGAATCAGCTGTTGACTGTGAAAGTGTTCCTTCCATGCCCAGGGTTGCCTTCACAATTGGAGGCAAAGAGTTTGAGCTCGCTCCAGAGGAGTACATTCTCAAGGTTGGCCAGGGTTCAGCTGCCCAGTGCATCAGTGGCTTTACTGCTTTGGATATACCTCCTCCTCGTGGACCTCTCTGGATACTGGGAGATATTTTCATGGGTCGTTACCACACTGTCTTTGATTCTGGAAAGCTAAGAGTTGGATTTGCTGAGGCAGCTTAA
SEQ ID NO.2
MGVNLKAIVGFVFLSFLLFAVVSSASNDGLLRIGLKKVKLDKNNRIAARLDSKETLRASIRKYNLCSNLGESGDTDIVALKNYLDSQYYGEIGVGSPPQKFTVIFDTGSSNLWVPSSKCYLSVACYFHSKYDSGKSSTYKKNGKSAEISYGSGSISGFFSNDAIEVGGLVVKDQEFIEATKEPSITFLAAKFDGILGLGFKEISVGDAVPVWDNMIKHGLIKDPVFSFWLNRNAEDEEGGEIVFGGMDPKHYKGKHTFVPVTQKGYWQFNMGDVHIGDKPTGYCASGCAAIADSGTSLLAGPTTIITMINQAIGASGVVSQQCKAVVSQYGEAIMDLLLSEAQPKRICSQIGLCTFDGTRGISISIQSVVDEGNDKSSGVLGDAMCSACEMAVVWMRSQLKQNQTQDRILDYASQLCERMPNPMGESAVDCESVPSMPRVAFTIGGKEFELAPEEYILKVGQGSAAQCISGFTALDIPPPRGPLWILGDIFMGRYHTVFDSGKLRVGFAEAA
SEQ ID No.3
PagAPA1-F:
agaacacgggggactATGGGAGTGAACTTGAAAGCAATTG
SEQ ID No.4
PagAPA1-R:
acccccggggatcctAGCTGCCTCAGCAAATCCAACT
SEQ ID No.5
qPCR-APA1-F:AGTCATCTGGTGTTCTTGGTG
SEQ ID No.6
qPCR-APA1-R:TCTTGTGTCTGGTTCTGCTTC
Claims (10)
1.84K杨PagAPA1基因在调控树木次生木质部发育中的应用，其特征在于，所述84K杨PagAPA1基因的核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用，其特征在于，所述84K杨PagAPA1基因编码蛋白的氨基酸序列如SEQ ID No.2所示。
3.根据权利要求1所述的应用，其特征在于，所述应用是通过PagAPA1基因的过量表达使84K杨生长迟缓，茎干木质部宽度增加，茎干木质部面积占茎面积的比率增加，木质素合成增加。
4.根据权利要求3所述的应用，其特征在于，所述应用是利用PagAPA1基因构建植物表达载体，然后将含有PagAPA1基因的植物表达载体转入84K杨中，获得转基因植株，具体包括如下步骤：
(1)克隆84K杨PagAPA1基因，所述PagAPA1基因的核苷酸序列如SEQ ID No.1所示；
(2)将PagAPA1基因构建到植物表达载体中，获得重组载体；
(3)将步骤(2)获得的重组载体转化到84K杨中，获得转基因植株，并通过抗性筛选、鉴定，获得转基因阳性植株。
5.根据权利要求4所述的应用，其特征在于，步骤(1)所述84K杨为84K杨(Populus alba×Populus glandulosa)野生型植株。
6.根据权利要求4所述的应用，其特征在于，所述步骤(1)中用于克隆84K杨PagAPA1基因的上游引物和下游引物核苷酸序列分别如SEQ ID No.3和SEQ ID No.4所示。
7.根据权利要求4所述的应用，其特征在于，所述步骤(2)中植物表达载体为pBI121。
8.根据权利要求4所述的应用，其特征在于，所述步骤(3)中对获得的转基因阳性植株进行实时荧光定量PCR检测，上游引物和下游引物核苷酸序列分别如SEQ ID No.5和SEQ IDNo.6所示。
9.根据权利要求4所述的应用，其特征在于，步骤(3)所述转化方法为农杆菌转化法。
10.权利要求9所述含有84K杨PagAPA1基因的植物表达载体在影响树木次生木质部发育中的应用。
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