CN117587032A - 84K杨PagMYB4基因及其用途 - Google Patents
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Abstract
本发明公开84K杨PagMYB4基因及其用途，属于基因工程技术领域。所述84K杨PagMYB4基因的CDS区核苷酸序列如SEQ ID NO.1所示。本发明通过对84K杨PagMYB4基因的克隆和分析，并结合农杆菌介导的遗传转化技术在拟南芥中过表达PagMYB4对该基因功能验证，发现PagMYB4基因表达与植物抗旱密切相关。本发明为杨树耐旱育种与生产提供了理论依据和相关基因。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及84K杨PagMYB4基因及其在调控植株干旱胁迫耐受性方面的用途。
背景技术
干旱是一个长期存在的世界性难题，全球干旱半干旱地区约占陆地面积的35％，遍及世界60多个国家和地区。随着全球气候变暖加剧，干旱等极端事件的发生频率显著增加。而植物因为无法像动物一样自由移动去寻找水源，干旱胁迫对其影响更为显著。据报道，干旱胁迫不仅可以抑制植物正常的生长发育，甚至还可以通过影响其组织内次生代谢物质的含量从而改变植物的干旱耐受性。
在植物长期的进化过程中，植物获得了一系列的生物化学和分子机制以抵御干旱胁迫。其中植物通过关闭气孔、减缓生长、衰老和休眠等“减少干旱下水分和养分的消耗；植物还利用强大的根系、向水性以及C4和CAM光合途径等从土壤中获取水分和养分，以及活性氧清除是植物体内重要的耐性机制。杨树作为一种模式树种，是三北防护林的主体树种，但品种单一,在生长过程中及易受到干旱、盐碱等非生物胁迫的危害，很大程度上影响其生长，甚至导致植株死亡，对农林业生产造成重要的影响。研究林木抗旱分子机理和培育抗旱新品种对干旱土地利用、生态环境建设与恢复、林业经济效益提高具有重要意义。
发明内容
本发明的目的在于提供一种从杨树中克隆得到的参与杨树干旱胁迫响应的基因，将其应用到耐旱树种的育种和生产当中。
为实现上述目的，本发明基于转录组测序结果鉴定到一个参与杨树干旱耐受的基因，已课题组前期鉴定到的84K为材料，克隆了该基因全场CDS序列，相应核苷酸序列如SEQID NO.1所示，将其命名为PagMYB4.
PagMYB4基因全长CDS全长807bp，编码的蛋白质由269个氨基酸残基组成，氨基酸序列如SEQ ID NO.2所示。该蛋白质分子量为29.86KDa。
PagMYB4表达模式分析：通过荧光定量PCR技术分析，PagMYB4在叶片中高表达，尤以成熟叶中最高；在干旱胁迫条件下，84K中PagMYB4表达量随处理时间的延长逐渐提高，处理12h后达到最大，随后显著下降。
进一步，本发明利用农杆菌介导的遗传转化验证PagMYB4基因功能，过表达PagMYB4后拟南芥植株中PagMYB4的表达量显著上调，在8％PEG胁迫条件下生长的OE-PagMYB4植株的生物量显著高于对照植株。
进一步的，发现相较于野生型，过表达植株具有较高的APX。
本发明提供了所述的PagMYB4基因或与SEQ ID NO.1所示序列具有至少70％同源性且编码的蛋白在功能上等价的同源基因在调控林木对干旱胁迫耐受性方面的应用。
具体的，所述应用为利用生物技术手段使得林木的PagMYB4基因或同源基因过表达，进而提高作物对盐胁迫的耐受性。
进一步的研究表明，林木的PagMYB4基因或同源基因功能过表达后，减少其活性氧爆发。
进一步的，所述林木为杨树。杨树PagMYB4基因过表达受植株的耐旱性显著提高。
进一步的，利用农杆菌介导的遗传转化方法侵染拟南芥从而过表达PagMYB4基因。具体的，所述应用包括：首先将核苷酸序列如SEQ ID NO.1所示的PagMYB4基因片段插入到pBI121载体的XbaI位点间，构建重组载体pBI121:PagMYB4，再将重组载体转化农杆菌GV3101，然后侵染拟南芥，获得耐旱性增强的突变体植株。
本发明具备的有益效果。
本发明通过对杨树PagMYB4基因的克隆和分析，并结合农杆菌介导的遗传转化方法过表达PagMYB4基因对该基因功能验证，发现PagMYB4基因表达与杨树耐旱性密切相关。本发明为林木尤其杨树的耐旱育种与生产提供了理论依据和相关基因。
附图说明
图1为PagMYB4基因表达模式。
图2为利用农杆菌介导的遗传转化方法过表达PagMYB4后干旱胁迫对拟南芥植株生长的影响。
图3为利用农杆菌介导的遗传转化方法过表达PagMYB4后干旱胁迫对拟南芥植株活性氧爆发的影响。
图4转基因植株相对表达量
具体实施方式
下面结合以下实施例对本发明做进一步说明。以下实施例仅用于说明本发明，不用来限制本发明的适用范围。在不背离本发明精神和本质的情况下,对本发明方法、步骤或条件所做的修改或替换,均属于本发明的范围。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
本发明以课题组前期鉴定到的84K为材料,分析杨树响应盐胁迫相关基因PagMYB4,对阐明杨树响应干旱胁迫的分子机理和育种及生产具有重要意义。
实施例
实施例1PagMYB4基因CDS区克隆和分析
1、PagMYB4基因CDS区克隆
基于转录组测序结果鉴定到一个参与杨树耐旱性的基因。以课题组前期鉴定到的84K为材料，克隆了该基因全长CDS序列，相应核苷酸序列如SEQ ID NO.1所示，并将其命名为PagMYB4。
采用总RNA提取试剂盒提取84K叶片总RNA，DNaseI去除基因组DNA污染，随后利用PrimeScripffMII 1st Strand cDNA Synthesis Kit反转录成单链cDNA。根据Blast到的序列设计引物，采用高保真酶扩增，引物序列为：
pBI121-MYB4-F：agaacacgggggactATGGGAAGGTCTCCTTGCTGT pBI121-MYB4-R：acccccggggatcctTTTCATCTCCAAACCTCTATAATCC
将扩增出来的产物，连接pBI121载体，转化大肠杆菌DH5α，挑取单克隆测序。所述目标基因片段也可以通过人工合成获得。
2、PagMYB4基因序列分析该基因CDS全长807bp，编码269个氨基酸，该蛋白质分子量为29.86KDa。
实施例2PagMYB4表达模式分析
取正常培养基中生长一月的84K幼苗进行组织表达分析，分别为根、茎、叶、老叶、成熟叶和幼嫩叶。
提取不同组织样品总RNA，反转录呈cDNA后，利用SYBR绿色荧光酶复合物和7500PCR仪分析PagMYB4表达量(qRT-PCR)变化，引物序列为：
qPCR-MYB4-F：TGCTTACATTAGAACCCACGG
qPCR-MYB4-R：TGCCACGTTTAAGGTCAGG
组织表达分析结果显示，PagMYB4在叶片中的表达量最高，尤以成熟叶中的最高。
实施例3农杆菌介导的遗传转化方法验证PagMYB4基因功能
1、pBI121:PagMYB4载体构建
提取84K总RNA,去除基因组DNA污染后，反转录成cDNA。根据前期克隆的序列，设计特异性引物，扩增得到PagMYB4基因小片段。
引物序列为(小写字母为酶切位点):
pBI121-MYB4-F：agaacacgggggactATGGGAAGGTCTCCTTGCTGT pBI121-MYB4-R：acccccggggatcctTTTCATCTCCAAACCTCTATAATCC
将扩增到的产物连接到pBI121载体上，转化大肠杆菌DH5a,涂板，挑取阳性克隆测序，提取测序正确单克隆质粒转化农杆菌GV3101。
2、侵染拟南芥
(1)萌发与移苗
将拟南芥种在1/2MS培养基上，待第二片真叶长出时，从培养基上移到土壤中。
(2)剪去拟南芥已开花结荚组织
拟南芥生长至抽苔(由于节间伸长进入营养生长的丛生型植物的茎，受到温度和日照长度等环境变化的刺激，随着花芽的分化，茎开始迅速伸长，植株变高，常见于蒜苔)状态时，应剪掉茎组织，再生长一段时间，使整盘拟南芥全部抽苔，剪掉已经结种子留下花蕾，准备进行农杆菌侵染实验。
(3)农杆菌侵染液的制备
提前将阳性单克隆菌液大摇至OD600为2.0左右，用50ml离心管收集菌体后，倒掉上清，用5％蔗糖溶液重新悬浮起来，重复一次，调OD至0.8-1.0之间，加0.5％表面活性剂。
①将阳性农杆菌在LB(Rif+Kan)平板划线，28℃培养2-3天；挑单菌落，接种于4ml液体LB(Rif+Kan)培养基中，220rpm/min摇菌过夜(18h)；
②取过夜菌1ml，接种于100ml有两种抗生素的液体(Rif+Kan)LB培养液中，28℃暗培养条件下，220rpm/min培养过夜；OD600在2.0左右，倒入灭菌的50ml灭菌EP中，5000rpm，28℃离心8min，收集菌体，弃掉上清培养液。
*用转化渗透液中(100ml：5％蔗糖+50μl拟南芥侵染：silwet)悬浮收集的农杆菌菌体，使OD600值约为0.8(菌的活性最适合侵染)
(4)拟南芥侵染
将拟南芥的花蕾部位置于含有农杆菌渗透液的EP管中1min，侵染结束放到吸水纸上轻轻蘸去多余菌液，倒放，在黑暗条件下培养24h后正常培养。
(5)为提高拟南芥转化效率，可在3天至7天后再次进行拟南芥泡花侵染工作。待拟南芥盛花期过后，可将拟南芥植株绑在竹签上，方便后续种子收获。
(6)转化子鉴定
待拟南芥种子成熟收获后，将进行转基因拟南芥种子验证。
1、标记基因筛选：将收到的种子种植于含有卡那霉素的培养基上，筛选出抗性幼苗。
2、PCR检测：提取拟南芥基因组，进行PCR扩增，核酸电泳观察有无目的基因。
转基因拟南芥是常见的验证基因功能的一种技术手段，弥补了很多作物，如棉花生长周期长，植株高大，生长环境要求较高的缺陷。可以较短周期内，获得更多有效信息，为后续基因功能研究奠定基础。
实施例4拟南芥异源表达验证PagMYB4基因功能
实验共设两个处理：Control(正常营养土)和8％PEG(每周浇一次)
结果显示：8％PEG处理3周后，L1和L3株系的耐旱性显著高于野生型。与WT相比，过表达植株生物量显著增加且APX含量显著增加。
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
ATGGGAAGGTCTCCTTGCTGTGAAAAAGCTCATACAAACAA
AGGCGCATGGACTAAGGAAGAAGATGATCGCCTTATTGCTT
ACATTAGAACCCACGGTGAAGGTTGCTGGCGTTCACTTCCT
AAAGCTGCTGGCCTTCTAAGATGCGGCAAGAGCTGCAGAC
TTCGTTGGATCAACTATTTAAGACCTGACCTTAAACGTGGC
AATTTTACTGAAGAAGAAGATGAGCTCATTATCAAACTCCA
TAGTCTCCTCGGCAACAAATGGTCACTTATAGCCGGAAGGT
TACCAGGGAGAACAGATAATGAGATAAAGAATTATTGGAAC
ACACATATAAGAAGGAAGCTCTTGAATAGAGGCATAGATCC
TGCGACTCATAGGCCACTCAATGAACCAGCCCAAGAAGCTT
CAACAACAATATCTTTCAGCACTACTACCTCAGTTAAAGAA
GAGTCGTTGAGTTCTGTTAAAGAGGAAAGTAATAAGGAGA
AGATAATTAGCGCAGCTGCTTTTATATGCAAAGAAGAGAAA
ACCCCAGTTCAAGAAAGGTGTCCAGACTTGAATCTTGAACT
TAGAATTAGCCTTCCTTGCCAAAACCAGCCTGATCGTCACC
AGGCATTCAAAACTGGAGGAAGTACAAGTCTTTGTTTTGCT
TGCAGCTTGGGGCTACAAAACAGCAAGGACTGCAGTTGCA
GTGTCATTGTGGGTACTATTGGAAGCAGCAGTAGTGCTGGC
TCCAAAACTGGCTATGACTTCTTAGGGATGAAAAGTGGTGT
GTTGGATTATAGAGGTTTGGAGATGAAATGASEQ ID NO.2
MGRSPCCEKAHTNKGAWTKEEDDRLIAYIRTHGEGCWRSLP
KAAGLLRCGKSCRLRWINYLRPDLKRGNFTEEEDELIIKLHSL
LGNKWSLIAGRLPGRTDNEIKNYWNTHIRRKLLNRGIDPATH
RPLNEPAQEASTTISFSTTTSVKEESLSSVKEESNKEKIISAAAF
ICKEEKTPVQERCPDLNLELRISLPCQNQPDRHQAFKTGGSTS
LCFACSLGLQNSKDCSCSVIVGTIGSSSSAGSKTGYDFLGMKS
GVLDYRGLEMK。
Claims (8)
1.84K杨PagMYB4基因，其特征在于，该基因的CDS区核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的84K杨PagMYB4基因编码的蛋白质，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
3.CDS区核苷酸序列如SEQ ID NO.1所示的PagMYB4基因或与SEQ ID NO.1所示序列具有至少70％同源性且编码的蛋白在功能上等价的同源基因在调控杨树对干旱胁迫耐受性的应用。
4.如权利要求3所述的应用，其特征在于，所述应用为：利用生物技术手段使得杨树的PagMYB4基因或同源基因功能缺失，降低杨树对干旱胁迫的耐受性。
5.如权利要求4所述的应用，其特征在于，杨树的PagMYB4基因或同源基因缺失后，减少其APX的含量。
6.如权利要求4所述的应用，其特征在于，所述应用包括：利用农杆菌介导的遗传转化技术导致所述PagMYB4基因或同源基因的表达提高，从而获得干旱胁迫的耐受性增强的突变体植株。
7.如权利要求3所述的应用，其特征在于，所述物种为杨树或拟南芥。
8.如权利要求7所述的应用，其特征在于，所述应用包括：首先讲核苷酸序列SEQ IDNO.3所示的PagMYB4基因片段插入到pBI121载体的XbaI位点间，构建重组载体pBI121：PagMYB4，提取质粒转化农杆菌GV3101后侵染拟南芥，获得干旱耐受性增强的突变体植株。
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