CN113293170B - 调控木薯淀粉含量的基因MeTIR1及其应用 - Google Patents

调控木薯淀粉含量的基因MeTIR1及其应用 Download PDF

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CN113293170B
CN113293170B CN202110585684.6A CN202110585684A CN113293170B CN 113293170 B CN113293170 B CN 113293170B CN 202110585684 A CN202110585684 A CN 202110585684A CN 113293170 B CN113293170 B CN 113293170B
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丁泽红
胡伟
颜彦
铁韦韦
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Sanya Research Institute Chinese Academy Of Tropical Agricultural Sciences
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Abstract

本发明公开一种调控木薯淀粉含量基因MeTIR1及其应用，所述MeTIR1的CDS核苷酸序列如SEQ ID No.1所示，其MeTIR1蛋白的氨基酸序列如SEQ ID No.2所示；本发明准确鉴定了MeTIR1在调控淀粉含量上的功能，为提高木薯淀粉含量的遗传改良提供了理论依据和关键基因，具有重要的应用价值；本发明表明MeTIR1第一个外显子上的G/T等位变异与木薯淀粉含量有关。通过检测该基因型，有利于快速筛选高淀粉木薯种质资源，加快木薯淀粉改良育种进程。

Description

调控木薯淀粉含量的基因MeTIR1及其应用
技术领域
本发明涉及植物基因工程的技术领域，具体涉及一种调控木薯淀粉含量基因MeTIR1及其应用。
背景技术
木薯(Manihot esculenta Crantz)是大戟科木薯属多年生植物，其块根富含淀粉，是生产工业淀粉、乙醇燃料、和生物基材料的重要原料，在我国农业和工业生产中占有举足轻重的地位。多年来由于木薯良种化问题没有得到足够的重视(主要以茎秆进行无性繁殖)，木薯品种单一且退化严重、淀粉含量低，造成木薯加工企业经济效益不能最大化，极大地限制了木薯产业的发展。
木薯淀粉遗传改良要依赖于淀粉代谢关键基因的理解，植物淀粉代谢途径中关键酶较多，多数以基因家族的形式存在。关于淀粉代谢途径及其关键酶的作用在拟南芥和主要禾谷类植物中已经取得很大进展。然而，与它们相比，我们对木薯块根淀粉代谢的了解还非常有限(主要是基于拟南芥或马铃薯中同源基因的认识)，其关键基因和调控网络尚不清楚，严重制约了木薯淀粉遗传改良。而且，与其他薯类和禾谷类作物截然不同的是，木薯的块根不能进行育苗繁殖(即不需要分解块根中的淀粉为下一代繁殖提供能量)，因而在淀粉代谢上很可能具有不同的调控机制。
因此，在木薯中进行淀粉代谢的研究，进而发掘木薯块根淀粉代谢关键基因，不但可以丰富植物淀粉代谢调控理论，而且对培育高淀粉木薯品种也具有重要意义。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种调控木薯淀粉含量基因MeTIR1及其应用，将基因MeTIR1可用于木薯淀粉含量的遗传改良，培育高淀粉木薯品种意义重大。
为实现上述目的，本发明所提供了一种调控木薯淀粉含量基因MeTIR1，所述MeTIR1的CDS核苷酸序列如SEQ ID No.1所示，其MeTIR1蛋白的氨基酸序列如SEQ ID No.2所示。
本发明还提供了一种上述基因MeTIR1在培育木薯新品种中的应用。
本发明还提供了一种调控木薯淀粉含量基因MeTIR1分子标记，其核苷酸序列如SEQ ID No.3所示，该标记序列第20碱基为多态性位点G/T。
本发明的有益效果：
(1)本发明准确鉴定了MeTIR1在调控淀粉含量上的功能，为提高木薯淀粉含量的遗传改良提供了理论依据和关键基因，具有重要的应用价值；
(2)本发明表明MeTIR1第一个外显子上的G/T等位变异与木薯淀粉含量有关。通过检测该基因型，有利于快速筛选高淀粉木薯种质资源，加快木薯淀粉改良育种进程。
附图说明
图1为基因MeTIR1挖掘及功能SNP分析图；
图1A为木薯野生种和栽培种块根中的淀粉含量差异；
图1B为野生种-栽培种之间差异表达且受到人工选择的生长发育相关基因；
图1C为基因MeTIR1模型，竖直的虚线表示第一个外显子中存在的G/T突变的位置，该突变可导致非同义突变Arg/Met；
图1D为基因MeTIR1的G/T突变在野生种和栽培种中的频率；
图1E为基因MeTIR1的GG、GT、和TT等位变异的块根淀粉含量，n代表具有相同基因型的木薯样品数量；
图1F为高淀粉(GG)和低淀粉(GT)品种在种植350天后块根中MeTIR1等位基因的表达差异，n代表具有相同基因型的木薯样品数量；
图2为MeTIR1调控淀粉含量的功能分析图；
图2A为MeTIR1在转基因木薯叶片中的表达分析，木薯F1015和R72携带GG等位基因，4363和Baodao9-1携带GT等位基因。上述四个品种分别用pCAMBIA1304(超表达载体对照)，pCAMBIA1304::MeTIR1(超表达)，pTRV(RNAi载体对照)或pTRV::MeTIR1(RNA沉默，RNAi)进行瞬时过表达和沉默转化实验，表达量通过qRT-PCR检测，每个样品测定3个生物学重复，数据表示为平均值±标准差，**表示P<0.01；
图2B为上午8:30转基因木薯叶片中的淀粉含量；
图2C为下午8:30转基因木薯叶片中的淀粉含量,木薯幼苗种植于人工气候室中，设置温度为27℃，光照为14h(从上午8:00到晚上10:00)和黑暗为10h(从晚上10:00到第2天上午8:00)。每个样品测定3个生物学重复，数据表示为平均值±标准差，**表示P<0.01。
具体实施方式
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。
实施例1木薯野生种-栽培种转录组比较分析，初步筛选调控淀粉含量的候选基因
1.木薯从野生种到栽培种的驯化过程中，块根淀粉含量极显著增加(图1A)。因此，通过野生种-栽培种转录组比较分析可以初步筛选调控木薯淀粉含量的候选基因；
2.在农业农村部儋州木薯种质资源圃任意选取了3个野生种和8个栽培种的块根(种植350天)进行转录组比较分析：
1)采用TruSeq(Illumina)试剂盒提取木薯RNA，
2)采用琼脂糖凝胶电泳和Nanodrop检查RNA浓度和完整性；
3)文库构建和转录组测序在天津诺禾致源生物信息科技有限公司进行；
4)测序数据下机后去除接头序列和低质量reads，通过HISAT2v2.0.4将干净reads比对到SC205参考基因组；
5)基因表达水平由StringTie v1.3.4d使用默认参数计算。基因表达水平采用FPKM进行归一化处理；
6)使用DESeq2软件鉴定差异表达基因，差异表达基因被定义为FDR<0.01且倍数变化>2。
野生种-栽培种转录组比较分析共奠定了16个与生长发育相关的差异表达基因(图1B)，且它们位于野生种-栽培种选择区间内。这些基因是调控木薯块根淀粉含量的候选基因；其中Sc02g014200编码一个转运抑制反应蛋白，其功能尚未见报道，将其命名为MeTIR1；其CDS核苷酸序列如SEQ ID No.1所示；其MeTIR1蛋白氨基酸序列如SEQ ID No.2所示。
实施例2基于基因组重测序比较分析，挖掘MeTIR1功能性SNP位点
从农业农村部儋州木薯种质资源圃选取了299份木薯栽培种材料，取其嫩叶采用DNeasy Plant Mini试剂盒(Qiagen，北京)提取基因组DNA。每个样品均用5μg基因组DNA构建插入片段大小为500bp的pair-end文库。使用Illumina X-Ten平台对每个样品的150bp配对末端读数进行测序。14份木薯野生种重测序数据来自于NCBI-SRA数据库，登录号为PRJNA234391。
使用BWA mem v0.7.17程序将每个样品的测序读数比对到木薯SC205参考基因组。使用Samtools v1.9和Picard v1.94对比对结果进行排序和重复标记处理。删除低质量的reads后，单端和双端比对上的reads均采用GATK toolkit v3.5流程检测SNP。HaplotypeCaller模块用于建立包含SNP和Indel的原始基因型文件，并使用以下参数对其进一步过滤：“QUAL<2.0||QD<2.0||MQ<40.0||FS>60.0||MQRankSum<-12.5||ReadPosRankSum<-8.0-clusterSize 2-clusterWindowSize 5”和“QD<2.0||FS>200.0||ReadPosRankSum<-20.0”。使用SnpEff v3.6c软件对已识别的SNP和Indel进行注释。
基于野生种-栽培种基因组重测序数据，发现MeTIR1第一个外显子中存在G/T突变，可导致非同义突变Arg/Met(图1C)。与野生种相比，栽培种具有较高的GG等位基因频率和较低的GT或TT等位基因频率(图1D)。而且，与GT或TT等位基因相比，GG等位基因具有更高的淀粉含量(图1E)和基因表达量(图1F)。
因此，该G/T突变是MeTIR1调控木薯淀粉含量的功能性SNP位点，含有该功能性SNP位点的标记序列如SEQ ID No.3所示；
利用上述序列设计了PCR扩增引物，
F1:CTGCGTCATTGGTCTGCAGG
F2:CTGCGTCATTGGTCTGCAGT
R:GCGAGATCGTCGTCCGTCA
以待检测品种的DNA为模板，利用F1+R和F2+R引物进行PCR扩增，经琼脂糖凝胶电泳检测：
(1)如果F1+R电泳有条带而F2+R电泳无条带，则表明该检测品种为GG等位基因；
(2)如果F1+R电泳无条带而F2+R电泳有条带，则表明该检测品种为TT等位基因；
(3)如果F1+R和F2+R电泳均有条带，则表明该检测品种为GT等位基因。
实施例3
超表达和RNAi功能验证表明MeTIR1在调控淀粉含量上发挥重要作用
为了证明MeTIR1在调控淀粉含量上发挥重要作用，我们选取了4个代表性木薯品种(其中F1015和R72携带GG等位基因；4363和Baodao9-1携带GT等位基因)，同时开展超表达和RNAi功能验证。
为了过表达MeTIR1，先扩增MeTIR1的编码序列并将其插入pCAMBIA1304载体，随后将带有重组载体或pCAMBIA1304(对照)的农杆菌菌株(GV3101)注射到木薯叶片中。为了沉默MeTIR1，扩增MeTIR1特异性区域并将其克隆到pTRV2载体中，随后将带有重组载体或pTRV2(对照)的农杆菌菌株(GV3101)与pTRV1一起注射到木薯叶片中。待pCAMBIA1304::MeTIR1和pCAMBIA1304转化的植物培养两天，或者pTRV::MeTIR1和pTRV转化的植物培养两周后，收集木薯叶片通过qRT-PCR检测基因表达量，同时采用淀粉测定试剂盒(A148-1-1，南京)检测淀粉含量。结果显示：超表达植株中MeTIR1的表达量显著提高了，伴随着淀粉含量的显著上升；而RNAi植株中MeTIR1的表达量显著降低了，与此同时，其淀粉含量也显著减少了(图2)。
结果表明，MeTIR1在调控淀粉含量上发挥重要作用，可用于提高木薯淀粉含量的遗传改良。
其它未详细说明的部分均为现有技术。尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
序列表
<110> 中国热带农业科学院热带生物技术研究所
中国热带农业科学院三亚研究院
<120> 调控木薯淀粉含量基因MeTIR1及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1899
<212> DNA
<213> 木薯(Manihot esculenta)
<400> 1
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gaggatcgca ctgaaatgtc tgaagacgat gaccgatctc tgctttctga ttctgttacc 120
actactgatt ccggttctac ttccgggtct ggactcaatt ctgtacccga gttccaagct 180
ccttatcctg accaggtctt ggaaaatgtc ctcgagaacg ttctctgctt cttgacctct 240
cgccgcgata gaaacgctgc gtcattggtc tgcaggtctt ggtaccgtgc tgaggcgctc 300
actcgatctg aactctttat cggaaactgc tatgctgttt ctccgcgacg cgccacgtcg 360
cggttcaccc gaatccggtc cgtaacgttg aaggggaagc cccgtttcgc cgatttcaac 420
ctcatgccac ccaattgggg ggcccacttc gcgccttggg tttctgcgat ggctaaggcg 480
tacccttggc tggagaaggt tcacctgaag cgcatgaccg tgacggacga cgatctcgct 540
ctccttgcag aatcattttt cgggttcaag gaacttgtgc tgatgtgttg tgatggattc 600
ggtactagcg ggctcgctgt ggttgctagt aggtgcagac aactgagagt gcttgatctg 660
cttgaatctg aggtatcaga tgatgaagtt gattggatat cattttttcc agagagtgaa 720
atgtgtctcg aatctcttat ttttgattgc gtagaatgcc acatcaattt tgatgcattg 780
gagaggctga ttgcacggtc accttcactg aagaaactca ggttgaacag aaatgtttca 840
attggccagc tacaccgact gatggtacga gctccgcagc tcacacatct tggcacaggt 900
tcatttcgac catcagagga tgtggctgag ggtgaacaag aaccagatta cgtatctgca 960
ttttctgctt gtgaatcctt agtttgcctc tctggtttta gggacattgt tccagactac 1020
ttgccagcaa tatacccagt ttgtgccaat ctcacttctc tgaatttaag ctatgcgaat 1080
atcaatgcag aacagctcaa acccataata agcaattgcc ataagcttca gattttctgg 1140
gtacttgatt caatatgtga tgaaggactt caggctgtgg ctgcaacatg caaggaactc 1200
cgggagctgc gggtcttccc tattgatgcc cgggaggata gcgaaggccc tgtttctgaa 1260
gttggtctcc aagcgatttc agagggttgt agaaagcttc aatctatttt atatttttgc 1320
cagcggatga cgaatgcagc tgttatagca atgtcaaaga actgcccaga tcttgtggtg 1380
ttccgtcttt gtataatggg acgccacagg cctgatcata tcactggaga gcctatggat 1440
gaaggatttg gagccattgt catgaattgt aagaatctca ctcggcttgc tgtatcaggt 1500
ttattgactg atagagcttt cagttatatt ggaaaatatg gtaaaacagt aaggaccctg 1560
tctgttgctt ttgctgggga cagtgacatg gggctgaagt atgtgctaga gggctgcccc 1620
agattgcaga agcttgagat tagagatagt ccatttgggg atgcagcttt gctgtctggt 1680
ctgcatcact attacaacat gagattcctt tggatgtctt cctgtaagtt aacacctgct 1740
ggttgccagc agattgctcg agctttgccc cgtctggtag tggaagtgat taatcacgaa 1800
ctcgatgagg acatgggtaa ttttgttaat acattataca tgtatcgatc tcttgaaggg 1860
ccaagagatg atgcaccaaa gtttgtttcc atcttgtag 1899
<210> 2
<211> 632
<212> PRT
<213> 木薯(Manihot esculenta)
<400> 2
Met Val Ile Ser Lys Lys Pro Arg Ser His Asn His Thr Leu Asp Pro
1 5 10 15
Asn Phe Met Arg Glu Asp Arg Thr Glu Met Ser Glu Asp Asp Asp Arg
20 25 30
Ser Leu Leu Ser Asp Ser Val Thr Thr Thr Asp Ser Gly Ser Thr Ser
35 40 45
Gly Ser Gly Leu Asn Ser Val Pro Glu Phe Gln Ala Pro Tyr Pro Asp
50 55 60
Gln Val Leu Glu Asn Val Leu Glu Asn Val Leu Cys Phe Leu Thr Ser
65 70 75 80
Arg Arg Asp Arg Asn Ala Ala Ser Leu Val Cys Arg Ser Trp Tyr Arg
85 90 95
Ala Glu Ala Leu Thr Arg Ser Glu Leu Phe Ile Gly Asn Cys Tyr Ala
100 105 110
Val Ser Pro Arg Arg Ala Thr Ser Arg Phe Thr Arg Ile Arg Ser Val
115 120 125
Thr Leu Lys Gly Lys Pro Arg Phe Ala Asp Phe Asn Leu Met Pro Pro
130 135 140
Asn Trp Gly Ala His Phe Ala Pro Trp Val Ser Ala Met Ala Lys Ala
145 150 155 160
Tyr Pro Trp Leu Glu Lys Val His Leu Lys Arg Met Thr Val Thr Asp
165 170 175
Asp Asp Leu Ala Leu Leu Ala Glu Ser Phe Phe Gly Phe Lys Glu Leu
180 185 190
Val Leu Met Cys Cys Asp Gly Phe Gly Thr Ser Gly Leu Ala Val Val
195 200 205
Ala Ser Arg Cys Arg Gln Leu Arg Val Leu Asp Leu Leu Glu Ser Glu
210 215 220
Val Ser Asp Asp Glu Val Asp Trp Ile Ser Phe Phe Pro Glu Ser Glu
225 230 235 240
Met Cys Leu Glu Ser Leu Ile Phe Asp Cys Val Glu Cys His Ile Asn
245 250 255
Phe Asp Ala Leu Glu Arg Leu Ile Ala Arg Ser Pro Ser Leu Lys Lys
260 265 270
Leu Arg Leu Asn Arg Asn Val Ser Ile Gly Gln Leu His Arg Leu Met
275 280 285
Val Arg Ala Pro Gln Leu Thr His Leu Gly Thr Gly Ser Phe Arg Pro
290 295 300
Ser Glu Asp Val Ala Glu Gly Glu Gln Glu Pro Asp Tyr Val Ser Ala
305 310 315 320
Phe Ser Ala Cys Glu Ser Leu Val Cys Leu Ser Gly Phe Arg Asp Ile
325 330 335
Val Pro Asp Tyr Leu Pro Ala Ile Tyr Pro Val Cys Ala Asn Leu Thr
340 345 350
Ser Leu Asn Leu Ser Tyr Ala Asn Ile Asn Ala Glu Gln Leu Lys Pro
355 360 365
Ile Ile Ser Asn Cys His Lys Leu Gln Ile Phe Trp Val Leu Asp Ser
370 375 380
Ile Cys Asp Glu Gly Leu Gln Ala Val Ala Ala Thr Cys Lys Glu Leu
385 390 395 400
Arg Glu Leu Arg Val Phe Pro Ile Asp Ala Arg Glu Asp Ser Glu Gly
405 410 415
Pro Val Ser Glu Val Gly Leu Gln Ala Ile Ser Glu Gly Cys Arg Lys
420 425 430
Leu Gln Ser Ile Leu Tyr Phe Cys Gln Arg Met Thr Asn Ala Ala Val
435 440 445
Ile Ala Met Ser Lys Asn Cys Pro Asp Leu Val Val Phe Arg Leu Cys
450 455 460
Ile Met Gly Arg His Arg Pro Asp His Ile Thr Gly Glu Pro Met Asp
465 470 475 480
Glu Gly Phe Gly Ala Ile Val Met Asn Cys Lys Asn Leu Thr Arg Leu
485 490 495
Ala Val Ser Gly Leu Leu Thr Asp Arg Ala Phe Ser Tyr Ile Gly Lys
500 505 510
Tyr Gly Lys Thr Val Arg Thr Leu Ser Val Ala Phe Ala Gly Asp Ser
515 520 525
Asp Met Gly Leu Lys Tyr Val Leu Glu Gly Cys Pro Arg Leu Gln Lys
530 535 540
Leu Glu Ile Arg Asp Ser Pro Phe Gly Asp Ala Ala Leu Leu Ser Gly
545 550 555 560
Leu His His Tyr Tyr Asn Met Arg Phe Leu Trp Met Ser Ser Cys Lys
565 570 575
Leu Thr Pro Ala Gly Cys Gln Gln Ile Ala Arg Ala Leu Pro Arg Leu
580 585 590
Val Val Glu Val Ile Asn His Glu Leu Asp Glu Asp Met Gly Asn Phe
595 600 605
Val Asn Thr Leu Tyr Met Tyr Arg Ser Leu Glu Gly Pro Arg Asp Asp
610 615 620
Ala Pro Lys Phe Val Ser Ile Leu
625 630
<210> 3
<211> 283
<212> DNA
<213> 木薯(Manihot esculenta)
<400> 3
ctgcgtcatt ggtctgcagk tcttggtacc gtgctgaggc gctcactcga tctgaactct 60
ttatcggaaa ctgctatgct gtttctccgc gacgcgccac gtcgcggttc acccgaatcc 120
ggtccgtaac gttgaagggg aagccccgtt tcgccgattt caacctcatg ccacccaatt 180
ggggggccca cttcgcgcct tgggtttctg cgatggctaa ggcgtaccct tggctggaga 240
aggttcacct gaagcgcatg accgtgacgg acgacgatct cgc 283

Claims (2)

1.一种调控木薯淀粉含量基因MeTIR1在培育木薯新品种中的应用；其中，所述MeTIR1的CDS核苷酸序列如SEQ ID No.1所示。
2.一种调控木薯淀粉含量基因MeTIR1分子标记，其核苷酸序列如SEQ ID No.3所示，该标记序列第20碱基为多态性位点G/T。
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