CN113897373A - 珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用 - Google Patents
珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用 Download PDFInfo
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- CN113897373A CN113897373A CN202111212593.4A CN202111212593A CN113897373A CN 113897373 A CN113897373 A CN 113897373A CN 202111212593 A CN202111212593 A CN 202111212593A CN 113897373 A CN113897373 A CN 113897373A
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- poplar
- mzasmt1
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Abstract
本发明公开了珠眉海棠MzASMT1基因在调控杨树根系发育中的应用,属于植物基因工程技术领域。本发明包括:提取珠眉海棠植株总RNA,反转录后获得cDNA,并以PCR技术从珠眉海棠植株cDNA中扩增出MzASMT 1基因编码序列;将所获得MzASMT 1基因***载体pCAMBIA2300中,获得植物表达载体;将所得植物表达载体转化到根瘤农杆菌EHA105中;将所得农杆菌转化子侵染杨树叶片,获得转基因杨树植株。与野生型杨树相比,转基因杨树植株最长主根的长度、总侧根的长度及总根系的干重明显降低,表明MzASMT 1基因能有效调控杨树根系发育,本发明在杨树育种工作中具有重要应用价值。
Description
技术领域
本发明属于植物基因工程技术领域,更具体地说,涉及珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用。
背景技术
根是植物长期适应陆地生境而形成的一个重要器官,是土壤与植物地上部分之间物质交换的桥梁,是植物健康生长和抵御逆境的基础。根据发生部位不同,根可分为定根(主根和侧根)和不定根两大类。杨树根系发达,分布深而广远。成年杨树根系分布半径长达8-10米,深2米左右。即使在栽植株行距7-8米的林地,在0-80cm的土层中,它的根也到处都是。一棵七年生杨树,根系总重34.7千克,以其树高、胸径推算,根重约占全树(不含树叶)总重的七分之一左右。由于其根系多而粗,许多道路和建筑设施都被杨树根系破坏,因此,如何调控杨树的根系发育具有重要的意义。
ASMT属O-甲基转移酶OMT家族。ASMT最初从水稻中被克隆出来,在水稻叶片衰老和其他几种逆境下表达量提升,同时促进了内源褪黑素的合成。它与发现于动物中的羟基吲哚-O-甲基转移酶(HIOMT)高度同源,可能是植物褪黑素生物合成途径中的限速酶。褪黑素对植物的营养生长起着促进的作用,适宜浓度的褪黑素能提高植物韧皮部对光合产物的运输能力,将叶片的同化产物输送给整个植株,加强植物的营养生长;在浓度过高时,糖类进入韧皮部的过程受阻,不仅使整个植株接收的同化产物减少,积累在叶片的糖类还对光合作用产生了不利影响。通过转基因技术促进植物内源褪黑素的合成后,大部分转基因植株的生长都更加旺盛,根系也比野生型更加发达,有利于转基因植物更好的生长,应对各种生物胁迫和非生物胁迫。因此,通过基因工程技术获得根系发育适当的植物新品种具有很强的可操作性。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用:将珠眉海棠MzASMT 1基因转入杨树中表达,使杨树根系长度或重量比野生型低,所述珠眉海棠MzASMT 1基因的核苷酸序列如SEQ ID NO.1所示。
所述珠眉海棠MzASMT1基因在调控杨树根系发育中的应用,具体包括以下步骤:
1)构建珠眉海棠MzASMT 1基因的植物表达载体;
2)将所构建的珠眉海棠MzASMT 1基因的植物表达载体转入农杆菌;
3)使用步骤2)所得农杆菌侵染杨树组织或杨树细胞;
4)培育筛选杨树组织或杨树细胞,得到杨树根系长度或重量比野生型降低的转基因杨树。
进一步地,步骤1)中,所述珠眉海棠MzASMT 1基因的植物表达载体具体为pCAMBIA2300-MzASMT 1。
进一步地,步骤2)中,所述农杆菌为EHA105。
进一步地,步骤3)中,所述杨树组织具体为杨树叶片。
进一步地,步骤3)中,所述农杆菌侵染的具体方式为浸泡法。
相比于现有技术,本发明的有益效果为:
本发明将从珠眉海棠植株中克隆到的褪黑素生物合成相关基因MzASMT 1构建到农杆菌中,然后侵染野生型杨树,获得转MzASMT 1基因的转基因杨树,然后观察转MzASMT 1基因的转基因杨树,尤其是根系发育相关的性状,发现与野生型杨树相比,转基因杨树植株最长主根的长度、总侧根的长度及总根系的干重明显降低,表明MzASMT 1基因能有效调控杨树根系发育。因此,本发明获得了一种可以调控杨树根系发育的方法,可以解决目前杨树由于其根系多而粗,许多道路和建筑设施都被杨树根系破坏的问题,在杨树育种工作中具有重要应用价值。
附图说明
图1为过表达MzASMT 1转基因杨树GUS染色鉴定和PCR鉴定结果图,结果表明转MzASMT 1基因杨树为阳性株系;
图2为转MzASMT 1基因杨树与野生型杨树的根系对比图;
图3为转MzASMT 1基因杨树与野生型杨树最长主根的长度比较,结果表明过表达MzASMT 1转基因株系最长主根的长度显著低于野生型杨树(P<0.05);
图4为转MzASMT 1基因杨树与野生型杨树总侧根长度的比较,结果表明过表达MzASMT 1转基因株系总侧根长度显著低于野生型杨树(P<0.05);
图5为转MzASMT 1基因杨树与野生型杨树总根系的干重比较,结果表明过表达MzASMT1转基因株系总根系的干重显著低于野生型杨树(P<0.05)。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1:珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用
采用基因克隆技术获得目的基因,并将其构建过表达载体,然后采用农杆菌转化法转入杨树叶片中,具体步骤如下:
1、提取珠眉海棠植株总RNA,反转录后获得cDNA,并以PCR技术从珠眉海棠植株cDNA中扩增出MzASMT 1基因编码序列(SEQ ID NO.1);
2、将所获得MzASMT 1基因***载体pCAMBIA2300中,获得植物表达载体pCAMBIA2300-MzASMT 1;
3、将所得植物表达载体转化到根瘤农杆菌EHA105中;
4、将所得农杆菌转化子侵染杨树叶片,获得转基因杨树植株。
5、统计转MzASMT 1基因杨树、野生型杨树的最长主根长度、总侧根长度、根系干重,比较其与野生型杨树的差异。
步骤1的具体过程为:提取珠眉海棠植株总RNA,反转录后获得cDNA作为扩增模板,以如下核苷酸序列为引物:MzASMT 1-F:5′-GACTctgcaggtcgaATGGAGGGAGATGAAGCAAGAGAT-3′,MzASMT 1-R:5′-GATCtctagagtcgaKCTAAAGAAAAACTTCAATGAGGGATCTCAAAC-3′,进行PCR扩增,扩增条件为94℃3min;再进行30个循环,每个循环94℃30s,60℃30s,72℃1min;n;72℃终延伸10min;n;反应体系为20μL:cDNA模板1μL、上下游引物各0.4μL(10μM)、10×Buffer2μL、dNTPs 1.6μL、rTaq酶0.1μL,ddH2O14.5μL。PCR产物进行琼脂糖凝胶电泳,用凝胶提取试剂盒进行割胶回收,回收产物测定浓度后储存于-20℃,或直接进行酶切,连接等反应。
步骤2的具体过程为:通过PstI、Xbal两个限制性内切酶将步骤1所得基因MzASMT1扩增产物连接到过表达载体pCAMBIA2300(卡那霉素抗性)中获得植物表达载体pCAMBIA2300-MzASMT 1。
步骤3的具体过程为:(1)取-80℃保存的农杆菌感受态细胞于手心片刻待其部分融化,处于冰水混合状态时***冰中;
(2)取100μL感受态细胞加入0.01-1.0μg质粒,手指轻敲管底混匀,依次于冰上、液氮中、37℃水浴和冰浴中各静置5min;
(3)加入无菌LB液体培养基,于28℃度震荡培养2-3小时;
(4)6000rpm离心1min收菌,取50μL上清液轻轻吹打重悬菌块涂布于含100mg/L卡那霉素的LB平板上,倒置于28℃摇床上培养2-3天。
(5)阳性克隆检测:
挑取白色单菌落,接种在含有100mg/L卡那霉素的LB液体培养基中,250rpm,28℃摇菌培养4小时,用引物进行PCR检测阳性克隆,鉴定无误后,摇菌待用于转化杨树叶片。
步骤4的具体过程:将鉴定无误的农杆菌转化子pCAMBIA2300-MzASMT 1接种于30mL LB液体培养基中(含50mg/mL卡那霉素)在温度设置成28℃的摇床中培养直至OD600为0.5-0.8,离心收集菌体,用30mL转化培养基MSo重悬农杆菌,采用浸泡法转化杨树叶片,取鲜嫩叶片,切成小片状,浸泡在农杆菌培养基中两分钟左右。取出叶片,置于不含抗生素的MS固体培养基上28℃暗培养2-3天,2-3天后移到含有卡那霉素的MS分化培养基上等待分化,一周换一次培养基,等待叶片分化长出抗性芽后进行检测。
转基因植株鉴定:
(1)染色鉴定:剪取上述试管苗叶片0.2cm,置于GUS染液中,37℃染色12h以上。之后使用无水乙醇进行脱色。叶片出现蓝色则表明报告基因成功的整合到植物基因组中,该植物即为转基因植株(图1A)。
(2)PCR鉴定:采用宝日生物技术(北京)有限公司生产的PCR试剂盒,对染色成蓝色的植株进行转基因植株的PCR验证(图1B)。以如下核苷酸序列为引物:MzASMT 1-F:5′-ATGGAGGGAGATGAAGCAAGAGAT-3′,MzASMT 1-R:5′-CTAAAGAAAAACTTCAATGAGGGATCTCAAAC-3′,进行PCR扩增,扩增条件为94℃3min;再进行30个循环,每个循环94℃30s,60℃30s,72℃1min;72℃终延伸10min;反应体系为20μL:DNA模板1μL、上下游引物各0.4μL(10μM)、10×Buffer 2μL、dNTPs 1.6μL、rTaq酶0.1μL,ddH2O14.5μL。
步骤5的具体过程为:选取生根后60天后的转MzASMT 1基因杨树和野生型杨树组培苗,通过观察表型,测定最长主根的长度、总侧根的长度及总根系干重,验证MzASMT 1基因在杨树根系发育中的作用。结果表明,与野生型杨树相比,过表达MzASMT 1基因显著降低了杨树最长主根的长度、总侧根的长度及总根系的干重(图2~5),表明MzASMT 1基因在杨树根系发育中起到了重要的作用。
综上所述,本发明可以有效的降低杨树最长主根的长度、总侧根的长度及总根系的干重,表明MzASMT 1基因可以用于调控杨树根系发育的程度。
序列表
<110> 江苏省中国科学院植物研究所
<120> 珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用
<130> 100
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1077
<212> DNA
<213> Malus zumi Mats
<400> 1
atggagggag atgaagcaag agatttgttt ggagcacagt cccatttgta caagcatgta 60
ttcagcttca taacttctat gtcactcaag tgtgtagtgc agctcggcat accggacata 120
attgaccgcc acggacaacc cattactcta cctgacttgg tcacagcact tcagatccac 180
ccggctaaaa ccggtaacgt gcaccggctc atgcgcctca tggtgcactc cggcttcttt 240
gcacgaaaac aagttcctaa aaatcatgta gaagcagatg aaggagaaga agaggcttat 300
gatctcacac cgtcttctag gctcctcctg aaagacaagg tacccagctt gtctccgttc 360
gttgtggcga tgctcgatcc agctttcgca gcaccgtggc agttcctcgg aaactggttc 420
cgaggaaccg aggtcacgcc tttcgagagc gctcatggga tggggatttg ggaatacggg 480
gaaggaaacc ctgaattcaa cagtcttttc aacaaggcaa tggctagtga ttctggaatg 540
atgaacttgg tgattaaaga ttgcaagcca atctttgatg ggttgagttc attggttgat 600
gttggtggtg ggactggaaa agttgctagg attctctgtg acgctttccc tcaactgaaa 660
tgcacagttc ttgaacttcc acacgttgtt gctgatttgc cagacagtga gaatttgaag 720
tttgttggag gagacatgtt ccaggttatc cctccagcgg atgctgtttt cctcaagctg 780
actttgcatg ctctgagcga tgaggaatgc ttgaaggttt tgaagaaatg cagggaagca 840
attgcaagca atggccaagg gaaagttata atcatagaca tagtgataaa tgaagagaaa 900
gatgagcatg aaataaccga agcaaagctc ttgttcgatc tgcttatgat ggttgtggtc 960
actggaagag agaggagcga gaaagactgg aaaaagctct tcctggaggc tggtttcagc 1020
ggctacaagg tgacaccaat atttggtttg agatccctca ttgaagtttt tctttag 1077
<210> 2
<211> 358
<212> PRT
<213> Malus zumi Mats
<400> 2
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Gly Asn Val His Arg Leu Met Arg Leu Met Val His Ser Gly Phe Phe
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Val Thr Pro Phe Glu Ser Ala His Gly Met Gly Ile Trp Glu Tyr Gly
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Glu Gly Asn Pro Glu Phe Asn Ser Leu Phe Asn Lys Ala Met Ala Ser
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Asp Ser Gly Met Met Asn Leu Val Ile Lys Asp Cys Lys Pro Ile Phe
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Ala Arg Ile Leu Cys Asp Ala Phe Pro Gln Leu Lys Cys Thr Val Leu
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Glu Leu Pro His Val Val Ala Asp Leu Pro Asp Ser Glu Asn Leu Lys
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Phe Val Gly Gly Asp Met Phe Gln Val Ile Pro Pro Ala Asp Ala Val
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Phe Leu Lys Leu Thr Leu His Ala Leu Ser Asp Glu Glu Cys Leu Lys
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Val Leu Lys Lys Cys Arg Glu Ala Ile Ala Ser Asn Gly Gln Gly Lys
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Val Ile Ile Ile Asp Ile Val Ile Asn Glu Glu Lys Asp Glu His Glu
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Ile Thr Glu Ala Lys Leu Leu Phe Asp Leu Leu Met Met Val Val Val
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Claims (6)
1.一种珠眉海棠MzASMT 1基因在调控杨树根系发育中的应用,其特征在于,将珠眉海棠MzASMT 1基因转入杨树中表达,使转基因杨树根系长度或重量比野生型低,所述珠眉海棠MzASMT 1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,包括以下步骤:
1)构建珠眉海棠MzASMT 1基因的植物表达载体;
2)将所构建的珠眉海棠MzASMT 1基因的植物表达载体转入农杆菌;
3)使用步骤2)所得农杆菌侵染杨树组织或杨树细胞;
4)培育筛选杨树组织或杨树细胞,得到杨树根系长度或重量比野生型降低的转基因杨树。
3.根据权利要求2所述的应用,其特征在于,步骤1)中,所述珠眉海棠MzASMT 1基因的植物表达载体具体为pCAMBIA2300-MzASMT 1。
4.根据权利要求2所述的应用,其特征在于,步骤2)中,所述农杆菌为EHA105。
5.根据权利要求2所述的应用,其特征在于,步骤3)中,所述杨树组织具体为杨树叶片。
6.根据权利要求5所述的应用,其特征在于,步骤3)中,所述农杆菌侵染的具体方式为浸泡法。
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