CN117568396A - 过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用 - Google Patents
过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用 Download PDFInfo
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Abstract
本发明公开了过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用,通过从野生型毛白杨中克隆到毛白杨独脚金内酯受体PtoD14基因,采用基因工程方法将PtoD14基因导入毛白杨植株中,获得了次生发育增强的毛白杨株系,3月龄的转PtoD14基因毛白杨中次生木质部细胞层数,比3月龄的非转化普通毛白杨多了8‑21层,该发明对于为毛白杨规模化生产提供高木材产量具有重要意义。
Description
技术领域
本发明涉及生物技术领域,具体涉及过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用。
背景技术
木材是世界上最丰富的可再生资源之一,是天然环保和低能耗材料。木材的形成是一个复杂的发育过程,分为初生生长和次生生长两个阶段。木本植物在初生生长阶段,顶端分生组织中的干细胞不断产生新的细胞,支持植物向上生长同时产生侧枝和叶等器官(Aichinger et al.,2012;Miyashima et al.,2013;Ohashi-Ito and Fukuda,2010;Weigel and Jurgens,2002)。而次生生长阶段植物则借助形成层不断向内外两侧生成的次生木质部和次生韧皮部的不断分裂进行垂直于初生生长的次生生长,从而使植物进行横向生长,使茎干加粗(Chao et al.,2018)。次生生长是多年生木本植物木材形成的生物学基础,次生木质部的发育机制不仅是提高木材产量、改善木材品质的理论基础,也是解析遗传和环境因素协同调控的理想模型。但传统林木育种存在周期长、选育难等多种困难。因此,利用基因工程技术创制此生维管组织高度发育的林木新种质具有广阔的发展前景。
杨树(Populus spp.)是世界上中纬度平原地区栽培面积最大的速生用材树种之一,具有生长快、产量高和易于更新的特点,在木材、造纸以及防风固沙等方面广泛应用和种植。此外,杨树根深枝茂,能够防风固沙,减少水土流失,是营造防林及城乡绿化的理想树种(施恭明等.,2009)。除了重要的生态价值外,杨树也具有广泛的工业和建筑用途,杨树可以用于制浆造纸、是纤维板、胶合板的原材料,也被作为建筑和家具用材,同时是生物能源产业的重要原料(吴定新等.,1997;傅峰等.,1999;黄北等.,2013)。
毛白杨是我国特有的乡土树种,因其生长迅速、材质优良、适应性强的特点也成为北方地区广泛种植的用材树种。近年来,毛白杨遗传转化体系和基因敲除技术的建立使深入研究其木材形成和环境适应等重要性状的调控机理成为可能(Fan et al.2015;Xu etal.2017)。随着对杨树次生发育调控机制的深入研究和解析,如果通过基因工程技术创制促进杨树次生发育的优良树种进而提高木材产量具有重要的科学意义和经济价值。
植物激素独脚金内酯(SLs)被报道对于形成层活性具有正向调节作用(Agusti etal.,2011)。作为植物生长发育的关键调节因子,DWARF14(D14)在独脚金内酯合成的下游起作用,充当激素信号的组成部分或将独脚金内酯转化为生物活性形式的酶。在拟南芥中,AtD14可以通过介导GR24的水解,进而调控下胚轴的生长(Yao R et al.,2016)。这些研究表明,D14在调控木本植物次生发育中具有重要作用,但其机制尚不清楚。
发明内容
有鉴于此,本发明的目的之一在于提供一种过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用;本发明的目的之二在于提供一种提高木材产量的方法。
为达到上述目的,本发明提供如下技术方案:
1、过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用,所述独脚金内酯受体PtoD14基因的序列如SEQ ID NO.1所示。
本发明优选的,所述木材为毛白杨。
本发明优选的,所述提高木材产量是通过增强株高和茎粗。
本发明优选的,所述提高木材产量是通过增加木质部层数。
2、一种提高木材产量的方法,将毛白杨独脚金内酯受体PtoD14基因采用基因工程方法导入毛白杨植株中,获得超量表达毛白杨独脚金内酯受体PtoD14基因的植株,即为木材产量提高的毛白杨;所述独脚金内酯受体PtoD14基因的序列如SEQ ID NO.1所示。
本发明优选的,所述毛白杨独脚金内酯受体PtoD14基因由木质部特异性表达的启动子LMX5pro驱动表达,所述木质部特异性启动子LMX5pro的核苷酸序列如SEQ ID NO.3所示。
本发明优选的,所述基因工程方法是将含有PtoD14基因的重组载体通过农杆菌介导导入毛白杨植株中。
本发明优选的,所述含有PtoD14基因的重组载体是将SEQ ID NO.1所示的核苷酸序列连入pCAMBIA1300载体的BamH I和Sac I酶切位点处而得。
本发明的有益效果在于:本发明公开了过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用,通过从野生型毛白杨中克隆到毛白杨独脚金内酯受体PtoD14基因,采用基因工程方法,将PtoD14基因导入毛白杨植株中,获得了次生发育增强的毛白杨株系,3月龄的转PtoD14基因毛白杨中次生木质部细胞层数,比3月龄的非转化普通毛白杨多了8-21层,该发明对于为毛白杨规模化生产提供高木材产量具有重要意义。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为毛白杨LMX5-PtoD14特异性过表达阳性植株鉴定(A:野生型和LMX5-PtoD14超表达阳性株系PCR鉴定结果;B:野生型和LMX5-PtoD14超表达阳性株系qPCR鉴定结果);
图2为特异性超表达PtoD14株系的宏观表型(A:野生型和LMX5-PtoD14超表达植株宏观表型;B野生型和LMX5-PtoD14超表达植株株高及茎粗统计)。
图3为特异性超表达PtoD14株系的切片分析(A:野生型和LMX5-PtoD14超表达植株切片表型分析;B:野生型和LMX5-PtoD14超表达植株木质部层数统计)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1、毛白杨PtoD14基因启动子的克隆
(1)毛白杨基因组总RNA的提取
用DEPC水将RNA提取所需要使用的药勺、研钵、研杵等浸泡过夜,高温高压灭菌后烘干备用。RNA提取试剂采用Axygen公司的试剂盒说明书进行操作,将所得RNA保存至-80℃冰箱备用。具体步骤如下:
1)将取下的新鲜植物组织,迅速用锡箔纸包裹投入液氮中速冻;
2)在去RNA酶的研钵中,用液氮将样本充分研磨成粉;
3)将粉末转移至AG buffer中,充分震荡混匀至匀浆状,室温静置5-10min;
4)4℃冷冻离心,12000rpm/min,10min;
5)取上清至新的1.5mL EP管中,精确估算上清体积加入0.5倍的无水乙醇,混匀;
6)将混合液转移至spin clum提取柱中,离心12000rpm/min,1min;
7)弃收集管中废液,在柱子中加入500μL PG buffer,离心12000rpm/min,1min;
8)弃废液,在柱子中加入600μL Wash buffer,离心12000rpm,30s;
9)重复步骤8)一次;
10)弃废液,将空管离心12000rpm/min,1min,除尽滤膜上的液体;
11)在膜中央加入30-50μL RElution buffer,室温静置2min,离心12000rpm/min,1min,得到总RNA;
12)取1μL RNA样品跑琼脂糖凝胶电泳,根据28s和18s观察RNA条带完整性检测提取质量。
(2)毛白杨PtoD14基因的克隆
提取毛白杨基因组总RNA,将所获的毛白杨基因组总RNA通过反转录酶反转录获得第一链cDNA,根据SEQ ID NO.1所示的DNA序列,设计并合成完整编码框的上游引物和下游引物,具体如下:
上游引物PtoD14-F:5'-GGATCCATGAGTAGCCTCATCCTAG-3'(SEQ ID NO.4);
下游引物PtoD14-R:5'-TCTAGATCACCGGGAAAGGGCTCGC-3'(SEQ ID NO.5);
以反转获得的第一链cDNA为模板,以SEQ ID NO.4所示上游引物和SEQ ID NO.5所示下游引物为引物对,经PCR扩增后进行测序验证,验证序列正确,编码的氨基酸如SEQ IDNO.2所示。
实施例2、毛白杨LMX5pro启动子的克隆
(1)毛白杨基因组基因组DNA的提取
采用改良的CTAB法提取杨树DNA,方法如下:
1)取1g新鲜野生型毛白杨叶片于研钵中,在液氮中研磨成粉;
2)加入3ml 1%的CTAB和90μL 65℃预热的β-巯基乙醇,65℃水浴30min,取出放至室温;
3)加入与CTAB等体积的氯仿:异戊醇(V/V为24:1),剧烈震荡后平放,乳化10min;
4)室温条件下,10000r/min,离心10min;
5)取上清于另以离心管中,重复3-4步;
6)取上清1ml到2ml离心管中,加入等体积预冷的异丙醇,轻轻晃动至出现絮状沉淀,4℃,10000r/min,离心10min;
7)用75%的乙醇漂洗沉淀两次;100%的乙醇漂洗一次,在37℃烘箱中烘干;
8)加入50μL的ddH2O,1μL RNase,37℃酶解处理1h;于-20℃保存。
(2)毛白杨LMX5pro启动子的克隆
提取毛白杨基因组总DNA,根据SEQ ID NO.3所示的DNA序列,设计并合成完整编码框的上游引物和下游引物,具体如下:
上游引物PtoLMX5-F:5'-GGAATTCCAATGTGGGCCTGGTGTTATAAAG-3'(SEQ ID NO.6);
下游引物PtoLMX5-R:5'-GGGTACCCGGTTGGTGGGGAAAGATGCATC-3'(SEQ ID NO.7);
以反转获得的第一链cDNA为模板,以SEQ ID NO.6所示上游引物和SEQ ID NO.7所示下游引物为引物对,经PCR扩增后进行测序验证。
实施例3、构建含PtoD14基因的重组植物表达载体及工程菌
将扩增后的LMX5pro通过连接酶连接到EocR I和Kpn I酶切后的pCAMBIA1300线性化载体上,并转化大肠杆菌DH5α,筛选阳性克隆,然后提取质粒做PCR检测和酶切验证,得到含有LMX5pro启动子的重组植物表达载体,命名为pCAMBIA1300-LMX5pro载体。由北京擎科生物科技股份有限公司测序确认基因的正确性。
进一步地,将扩增后的PtoD14基因(其DNA序列如SEQ ID NO.3所示)通过连接酶连接到BamH I和Xba I酶切后的pCAMBIA1300-LMX5pro线性化载体中。并转化大肠杆菌DH5α,筛选阳性克隆,然后提取质粒做PCR检测和酶切验证,得到含有LMX5pro启动子驱动PtoD14基因的重组植物表达载体,命名为pCAMBIA1300-LMX5pro:PtoD14载体。由北京擎科生物科技股份有限公司测序确认基因的正确性。
将pCAMBIA1300-LMX5pro:PtoD14载体转化根癌农杆菌GV3101,筛选阳性克隆,获得含pCAMBIA1300-PtoD14载体的工程菌,命名为GV3101-pCAMBIA1300-LMX5pro:PtoD14。
实施例4、根癌农杆菌介导PtoD14基因转化毛白杨
(1)农杆菌的两次活化培养
1)将GV3101-pCambia1300-LMX5pro:PtoD14菌种于含有40mg/L利福平和50mg/L卡那霉素的YEP固体培养基划线,28℃恒温培养箱中培养36h;挑取单菌落接种于至10mL YEP+Rif+kan的双抗液体培养基;
2)28℃,200rpm/min振荡培养36-48小时,使菌液浓度达到OD600=0.8-1.0;
3)按照1:1000的比例,吸取50μL一活菌液至50mL新鲜YEP+Rif+kan的双抗液体培养基中,进行二活液培养;
4)28℃,200rpm/min振荡培养12-16小时,使菌液浓度达到OD600=0.3-0.4,待用。
(2)农杆菌侵染液制备
1)利用50mL离心管收集二活菌液,4000rpm/min,8min,收集菌体;
2)弃去培养基上清液,利用25mL含有AS的WPM重悬液,将农杆菌重悬,重悬液倒入无菌的玻璃瓶中;
3)重悬液置于28℃,200rpm/min振荡1-2小时,使农杆菌侵染活性增强。
(3)叶盘制备
1)在超净台中,将灭过菌的剪刀、镊子、手术刀柄用酒精灯外焰灼烧15秒,放凉备用;
2)用剪刀将健康的野生型组培苗叶片剪下5-6片,放入培养皿中,皿中加入1/3体积的无菌水,使叶片保持湿润;
3)将无菌手术刀片装入刀柄,火焰灼烧后静置冷却,用刀片将叶片均匀切割成0.5cm2的正方形叶盘。
(4)侵染
1)将叶盘用镊子夹入农杆菌重悬液中,轻轻晃动玻璃瓶使重悬液均匀包裹叶盘,侵染10min;
2)侵染结束后,用镊子小心的将叶盘夹出,放到无菌纸上,吸干叶盘上多余的侵染液;
3)把叶盘平整的贴于共培养平板上,置于暗盒中,25℃暗培养36-48h。
(5)叶盘的选择培养
1)暗培养结束后,根据载体选择合适的植物抗性,配制含抗生素的选择培养基;
2)在超净台中将叶盘转移至选择培养基上,诱导愈伤组织。期间每七天,将叶盘更换至新的培养基上,持续更换3-4周,至叶盘边缘长出白色或淡黄色的愈伤组织。整个过程于暗盒中25℃培养。
(6)愈伤组织诱导生芽
将长出愈伤组织的叶盘转移至含对应抗生素的生芽培养基上,8000Lux,25℃光照培养5-6周,每周更换一次培养基。期间愈伤组织会充分生长膨大,第5周左右,愈伤组织上会长出芽点,生长出丛生芽。
(7)丛生芽诱导生根
当丛生芽长至约3-5cm,用锋利的剪刀将芽剪下,用镊子将丛生芽插入生根培养基中,8000Lux,25℃光照培养7-10天即可获得生根幼苗。此即为候选转基因植株,等待后续鉴定阳性后方可移栽土培,将转基因幼苗命名为LMX5-PtoD14植株。
实施例5、pCAMBIA1300-LMX5pro:PtoD14转基因植株PCR分子鉴定
(1)野生型和pCAMBIA1300-LMX5pro:PtoD14转基因毛白杨的DNA提取
分别选取10~15株转基因的抗性再生植株,提取毛白杨基因组DNA。方法如下:
1)准备CTAB缓冲液于65℃水浴锅预热备用;
2)取约0.5g野生型和pCAMBIA1300-LMX5pro:PtoD14转基因毛白杨的叶片,于液氮中磨成粉末,加入上述预热的CTAB抽提液500μL中,混匀;
3)于65℃水浴45min,中途间隔(轻柔)振荡三次混匀。
4)水浴结束后,冷却至室温,加入等体积氯仿:异戊醇(24:1),轻柔颠倒混匀后平放乳化10min,4℃,12000rpm/min,离心10min;
5)吸取上清于新的无菌离心管中,加入等体积-20℃预冷的异丙醇,颠倒混匀能见白色絮状沉淀;
6)4℃,12000rpm/min,离心10min,去上清液,用500ml 75%(V/V)乙醇漂洗沉淀两次,500ml无水乙醇再漂洗一次,去液体;将沉淀于37℃旋转蒸发仪中干燥至出现半透明;
7)用25μL无菌水溶解沉淀,获得野生型和pCAMBIA1300-LMX5pro:PtoD14转基因毛白杨叶片DNA粗提物;
8)向DNA粗提物中加入约1μl RNase,于37℃酶解RNA 1h;
9)将DNA样品放置于-20℃冰箱保存备用。
(2)阳性植株PCR扩增
由于野生型植株不含外源转入的pCAMBIA1300载体序列,故利用载体pCAMBIA1300-F和pCAMBIA1300-R引物扩增进行阳性植株筛选。以野生型DNA模板作为阴性对照,转基因植株对应的测序正确的载体质粒为阳性对照,对转基因植株的DNA进行PCR扩增及凝胶电泳成像,以鉴定转基因阳性株系(图1中A)。发现仅在pCAMBIA1300-LMX5pro:PtoD14质粒DNA和L1、L3和L5-L9的DNA中扩增出750bp大小的目的条带。证明PtoD14-OE L1、L3和L5-L9植株中成功转入了pCAMBIA1300-PtoD14。
利用载体pCAMBIA1300-F和基因引物pCAMBIA1300-R引物扩增进行筛选。设计的特异性引物,序列如下:
pCAMBIA1300-F:5′-ATGGTGAGCAAGGGCGAGGAGC-3′(SEQ ID NO.8);
pCAMBIA1300-R:5′-ACTTTATTGCCAAATGTTTGAACG-3′(SEQ ID NO.9)。
PCR反应体系同表2,反应程序:94℃预变性3min,1个循环;94℃变性30s,退火30s,72℃延伸1min,共31个循环;72℃延伸10min,1%琼脂糖凝胶电泳检测扩增产物。
PCR反应的体系如表1所示。
表1、PCR反应的体系
(3)阳性植株RT-qPCR鉴定
对WT和LMX5-PtoD14超表达植株的茎取材,提取RNA,反转录为cDNA。使用Takara的定量酶进行荧光定量PCR,反应体系如表2所示,扩增引物如下:
qPCR-PtoD14-F:5′-GATTCTCATCGGAGCATCA-3′(SEQ ID NO.10)
qPCR-PtoD14-R:5′-TACATCCTTAGATGTCTGG-3′(SEQ ID NO.11)。
如图1中B所示。结果显示,株系L6-L9中PtoD14基因的表达量显著增高。其中L6和L8株系被用于进一步表型分析。
表2、qPCR体系
使用德国耶拿荧光定量基因扩增仪进行扩增,qPCR程序为:95℃预变性3min;95℃,30sec,60℃,1min,60℃,30sec;95℃,15sec,循环40次。
实施例6、毛白杨PtoD14过表达植株表型分析
将一月龄组培苗移栽至花盆,于25℃长日照条件(16小时光照/8小时黑暗,光强10000lux)的温室中生长三个月。对WT、LMX5-PtoD14转基因杨树的株高和茎粗进行测定和统计。
结果如图2中所示,与WT植株相比LMX5-PtoD14 L6和L8植株的株高和茎粗都显著增强(图2)。
实施例7、LMX5-PtoD14转基因植株次生发育分析
对3月龄WT、PtoD14-OE转基因杨树进行切片观察。结果如图3中所示,在木质部中过表达PtoD14基因后,毛白杨次生木质部层数显著增多。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (8)
1.过量表达毛白杨独脚金内酯受体PtoD14基因在提高木材产量中的应用,其特征在于:所述独脚金内酯受体PtoD14基因的序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于:所述木材为毛白杨。
3.根据权利要求1所述的应用,其特征在于:所述提高木材产量是通过增强株高和茎粗。
4.根据权利要求1所述的应用,其特征在于:所述提高木材产量是通过增加木质部层数。
5.一种提高木材产量的方法,其特征在于:将毛白杨独脚金内酯受体PtoD14基因采用基因工程方法导入毛白杨植株中,获得超量表达毛白杨独脚金内酯受体PtoD14基因的植株,即为木材产量提高的毛白杨;所述独脚金内酯受体PtoD14基因的序列如SEQ ID NO.1所示。
6.根据权利要求5所述的方法,其特征在于:所述毛白杨独脚金内酯受体PtoD14基因由木质部特异性表达的启动子LMX5pro驱动表达,所述木质部特异性启动子LMX5pro的核苷酸序列如SEQ ID NO.3所示。
7.根据权利要求5所述的方法,其特征在于:所述基因工程方法是将含有PtoD14基因的重组载体通过农杆菌介导导入毛白杨植株中。
8.根据权利要求5所述的方法,其特征在于:所述含有PtoD14基因的重组载体是将SEQID NO.1所示的核苷酸序列连入pCAMBIA1300载体的BamH I和Sac I酶切位点处而得。
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