CN109576285A - 李PsMYB18基因及其编码蛋白与应用 - Google Patents

李PsMYB18基因及其编码蛋白与应用 Download PDF

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CN109576285A
CN109576285A CN201811595512.1A CN201811595512A CN109576285A CN 109576285 A CN109576285 A CN 109576285A CN 201811595512 A CN201811595512 A CN 201811595512A CN 109576285 A CN109576285 A CN 109576285A
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psmyb18
gene
lee
leu
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方智振
叶新福
周丹蓉
姜翠翠
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Pomology Research Institute Fujian Academy of Agricultural Sciences
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Abstract

本发明属于植物分子生物学领域，具体涉及一种李PsMYB18基因及其编码蛋白与应用。所述李PsMYB18基因基因的核苷酸序列为SEQ ID NO:1；基因编码的氨基酸序列为SEQ ID NO:2；含有李PsMYB18基因的重组表达载体为pCAMBIA1302‑PsMYB18；含有李PsMYB18基因的基因工程菌为GV3101‑PsMYB18。将含有PsMYB18基因的工程菌菌液注射入烟草叶片中，可显著抑制花色苷合成激活因子的花色苷诱导作用。

Description

李PsMYB18基因及其编码蛋白与应用
技术领域
本发明属于植物分子生物学领域，具体涉及一种李PsMYB18基因及其编码蛋白与应用。
背景技术
李是蔷薇科李属植物，为多年生落叶果树，广泛分布于世界各国，是我国重要的传统果树之一，我国李栽培面积和产量均居世界首位。李果实富含类胡萝卜素、维生素和花色苷等生物活性物质，具有预防多种疾病的功效，深受消费者喜爱。
过去的数十年中，在国内外李育种家通过杂交、实生或芽变选种等传统手段获得了一大批优良的李品种。但随着人们生活水平的不断提高和消费习惯的改变，人们对李果实的要求呈现出多样化的需求，针对特定性状快速培育大量特色、多样的李新品种，成为推动产业转型升级与供给侧结构改革，不断满足消费者多样化需求的关键。
发明内容
本发明的目的在于提供一种李PsMYB18基因及其编码蛋白与应用。
为实现上述目的，本发明采用如下技术方案：
李PsMYB18基因，所述基因的核苷酸序列为SEQ ID NO:1。
所述基因编码的氨基酸序列为SEQ ID NO:2。
制备含有李PsMYB18基因的重组表达载体pCAMBIA1302-PsMYB18。
制备含有李PsMYB18基因的基因工程菌GV3101-PsMYB18。
进一步地，将李PsMYB18基因应用于基因工程育种培育李新品种中。
本发明的优点在于：
本发明提供一种来源于李的对花色苷合成有抑制作用的PsMYB18基因、编码蛋白及含有该基因的重组质粒和重组基因工程菌，本发明可应用于基因工程育种，用于培育花色苷含量改变的培育李新品种。
附图说明
图1为李PsMYB18基因对烟草叶片积累花色苷的影响。
具体实施方式
实施例1 李PsMYB18基因克隆
通过比较转录组学手段秋姬李’果皮中发现一个在秋姬李着色过程中表达量上升的基因，命名为PsMYB18。以红色‘秋姬李’果皮为材料进行RNA提取和cDNA合成。以特异引物对PsMYB18-F：ATGAGGAAACCCTGCTGCGATAAGC和
PsMYB18-R：CTTCCACCTGGACGATGAATATTC，进行PCR 扩增获得PsMYB18编码区序列。PCR反应体系为50μl，成分包括为：I-5TM2xHigh-Fidelity Master Mix 25μl，上下游引物(10μM)各2μl，cDNA 1μl，H2O 20μl。PCR程序为：90℃预变性3min，35个 循环的98℃15s，56℃15s和72℃ 20s，72℃ 5min。
PsMYB18基因序列如下：
ATGAGGAAACCCTGCTGCGATAAGCAAGACACAAACAGAGGAGCTTGGTCCAAGCAAGAAGACCTCAAGCTCATCGATTACATTCGCAAACATGGCGAGGGTTGTTGGCGTACCCTTCCTCAGGCTGCAGGCCTACTTCGTTGCGGTAAAAGCTGTAGGCTAAGATGGATAAACTATCTACGGCCAGACCTTAAAAGAGGAAACTTTGCTGAAGATGAAGAAGATCTCATCATTAAACTTCATGCACTCCTAGGCAACCGGTGGTCGTTGATTGCTGGGAGATTGCCGGGACGTACAGACAATGAAGTGAAGAACTATTGGAACTCTCATTTGAGAAGAAAACTTATAAGCATGGGAATAGATCCAAATAACCACCGACCGAACACCATTTATCTCCCTCGCCCTCATCATAAAAACTCACAAGCTGTTAGCAGCACTGCAAAATTGTCTGCAGGTTTGAAAACCCCAACGAATGATCAGCCAACAAGATCCGGTGGCAATAATTGTGACCAAGTATCGGACGGTACAAGTTGCTTAGAGGATGAATCTTGTGGCCATGAGCTGCCTGATTTAAACCTTGACCTCACAATGACGGCTCCTTTGTCTAATTCAGAACCTGAAAATCTCAAGGAGGAGCAAAATGTTATGAGTCTAAAATGTCATCGGAATTTGCCTCGTCCACAAATATCCTTCCTCTCTTGA
实施例2 含PsMYB18基因的重组质粒和重组基因工程菌的制备
利用特异引物对：ACGGGGGACTCTTGACATGAGGAAACCCTGCTGCGATAAGC (划线部分为NcoI酶切位点)和ATTCGAGCTGGTCACCTCAAGAGAGGAAGGATATT（划线部分为BstEII酶切位点），以实施例1中获得的产物为模板进行PCR反应，PCR反应体系为50μl，成分包括为：I-5TM2xHigh-Fidelity Master Mix 25μl，上下游引物(10μM)各2μl，模板DNA 1μl，H2O 20μl。PCR程序为：90℃预变性3min，35个 循环的98℃15s，56℃15s和72℃ 20s，72℃ 5min。。将获得的PCR产物回收后，采用Aidlab One Step Seamless Cloning kit将目的片段连接到经NcoI和BstEII酶切的pCAMBIA1302载体中，测序验证，获得含PsMYB18基因的重组质粒。然后将阳性质粒导入农杆菌菌株GV3101中，获得含PsMYB18基因的重组基因工程菌，即含有超量表达载体的农杆菌菌株GV3101-PsMYB18。
实施例3 基因功能验证
选择烟草叶片验证PsMYB18基因功能。
1、实验方法：将PsMYB10.1（Genbank登录号MK105923）和PsbHLH（Genbank登录号MH383068）分别连接到经NcoI和BstEII酶切的pCAMBIA1302载体中，获得重组质粒pCAMBIA1302-PsMYB10.1和pCAMBIA1302-PsbHLH，将阳性质粒导入农杆菌菌株GV3101中，获重组基因工程菌GV3101-PsMYB10.1和GV3101-PsbHLH。用注射缓冲液（10mM MES,10mMgCl2, 100μM乙酰丁香酮）将GV3101-PsMYB18、GV3101-PsMYB10.1和GV3101-PsbHLH重悬至OD600为0.8，并在室温条件下静置3h。
选取长势优良本氏烟草，以叶片中心叶脉为分界线，将含有pCAMBIA1302-PsMYB10.1和pCAMBIA1302-PsbHLH质粒的菌液等体积混合，取50μl，用无针头注射器注射在左侧叶片上；将含有pCAMBIA1302-PsMYB10.1、pCAMBIA1302-PsbHLH和pCAMBIA1302-PsMYB18的菌液等体积混合后，取50μl，用无针头注射器注射在右侧叶片上；烟草于14h:10h光暗周期、24℃和70％空气湿度的条件下培养6天。
2、实验结果：
分析结果如图1显示，注射含pCAMBIA1302-PsMYB10.1 + pCAMBIA1302-PsbHLH农杆菌的烟草叶片积累花色苷，着紫红色，而注射含pCAMBIA1302-PsMYB10.1 + pCAMBIA1302-PsbHLH +pCAMBIA1302-PsMYB18的烟草叶片不积累花色苷。
以上所述仅为本发明的较佳实施例，与PsMYB18的DNA序列具有90％以上同源性的DNA分子、由该蛋白衍生的蛋白的经过一个或几个氨基酸残基的改变蛋白质、含有PsMYB18基因的重组表达载体、表达盒、转基因细胞系、转基因植物组织或重组菌及其他依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建省农业科学院果树研究所
<120> 李PsMYB18基因及其编码蛋白与应用
<130> 6
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 702
<212> DNA
<213> 人工序列
<400> 1
atgaggaaac cctgctgcga taagcaagac acaaacagag gagcttggtc caagcaagaa 60
gacctcaagc tcatcgatta cattcgcaaa catggcgagg gttgttggcg tacccttcct 120
caggctgcag gcctacttcg ttgcggtaaa agctgtaggc taagatggat aaactatcta 180
cggccagacc ttaaaagagg aaactttgct gaagatgaag aagatctcat cattaaactt 240
catgcactcc taggcaaccg gtggtcgttg attgctggga gattgccggg acgtacagac 300
aatgaagtga agaactattg gaactctcat ttgagaagaa aacttataag catgggaata 360
gatccaaata accaccgacc gaacaccatt tatctccctc gccctcatca taaaaactca 420
caagctgtta gcagcactgc aaaattgtct gcaggtttga aaaccccaac gaatgatcag 480
ccaacaagat ccggtggcaa taattgtgac caagtatcgg acggtacaag ttgcttagag 540
gatgaatctt gtggccatga gctgcctgat ttaaaccttg acctcacaat gacggctcct 600
ttgtctaatt cagaacctga aaatctcaag gaggagcaaa atgttatgag tctaaaatgt 660
catcggaatt tgcctcgtcc acaaatatcc ttcctctctt ga 702
<210> 2
<211> 233
<212> PRT
<213> 人工序列
<400> 2
Met Arg Lys Pro Cys Cys Asp Lys Gln Asp Thr Asn Arg Gly Ala Trp
1 5 10 15
Ser Lys Gln Glu Asp Leu Lys Leu Ile Asp Tyr Ile Arg Lys His Gly
20 25 30
Glu Gly Cys Trp Arg Thr Leu Pro Gln Ala Ala Gly Leu Leu Arg Cys
35 40 45
Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp Leu
50 55 60
Lys Arg Gly Asn Phe Ala Glu Asp Glu Glu Asp Leu Ile Ile Lys Leu
65 70 75 80
His Ala Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro
85 90 95
Gly Arg Thr Asp Asn Glu Val Lys Asn Tyr Trp Asn Ser His Leu Arg
100 105 110
Arg Lys Leu Ile Ser Met Gly Ile Asp Pro Asn Asn His Arg Pro Asn
115 120 125
Thr Ile Tyr Leu Pro Arg Pro His His Lys Asn Ser Gln Ala Val Ser
130 135 140
Ser Thr Ala Lys Leu Ser Ala Gly Leu Lys Thr Pro Thr Asn Asp Gln
145 150 155 160
Pro Thr Arg Ser Gly Gly Asn Asn Cys Asp Gln Val Ser Asp Gly Thr
165 170 175
Ser Cys Leu Glu Asp Glu Ser Cys Gly His Glu Leu Pro Asp Leu Asn
180 185 190
Leu Asp Leu Thr Met Thr Ala Pro Leu Ser Asn Ser Glu Pro Glu Asn
195 200 205
Leu Lys Glu Glu Gln Asn Val Met Ser Leu Lys Cys His Arg Asn Leu
210 215 220
Pro Arg Pro Gln Ile Ser Phe Leu Ser
225 230
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<400> 3
atgaggaaac cctgctgcga taagc 25
<210> 4
<211> 24
<212> DNA
<213> 人工序列
<400> 4
cttccacctg gacgatgaat attc 24
<210> 5
<211> 41
<212> DNA
<213> 人工序列
<400> 5
acgggggact cttgacatga ggaaaccctg ctgcgataag c 41
<210> 6
<211> 35
<212> DNA
<213> 人工序列
<400> 6
attcgagctg gtcacctcaa gagaggaagg atatt 35

Claims (5)

1.李PsMYB18基因，其特征在于，所述基因的核苷酸序列为SEQ ID NO:1。
2.一种如权利要求1所述李PsMYB18基因，其特征在于，所述基因编码的氨基酸序列为SEQ ID NO:2。
3.含有权利要求1所述李PsMYB18基因的重组表达载体pCAMBIA1302-PsMYB18。
4.含有权利要求1所述李PsMYB18基因的基因工程菌GV3101-PsMYB18。
5.一种如权利要求1-4任一所述的李PsMYB18基因在基因工程育种培育李新品种中的应用。
CN201811595512.1A 2018-12-25 2018-12-25 李PsMYB18基因及其编码蛋白与应用 Pending CN109576285A (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100809736B1 (ko) * 2006-05-15 2008-03-05 대한민국 안토시아닌 생합성 억제 활성을 가진 ｍｙｂ６０ 유전자와상기 유전자가 도입된 형질전환 상추 및 그 제조방법
CN104498506A (zh) * 2014-12-15 2015-04-08 青岛农业大学 一种编码红肉桃myb10转录因子的dna序列及应用

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KR100809736B1 (ko) * 2006-05-15 2008-03-05 대한민국 안토시아닌 생합성 억제 활성을 가진 ｍｙｂ６０ 유전자와상기 유전자가 도입된 형질전환 상추 및 그 제조방법
CN104498506A (zh) * 2014-12-15 2015-04-08 青岛农业大学 一种编码红肉桃myb10转录因子的dna序列及应用

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HUI ZHOU 等: "Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation", 《NEW PHYTOLOGIST》 *
ZHOU,H. 等: "Prunus persica R2R3-MYB transcription factor (MYB18) mRNA, complete cds", 《GENBANK DATABASE》 *
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