CN110747207A - 一种水稻硝酸还原酶NIA3蛋白基因OsNia3及其应用 - Google Patents
一种水稻硝酸还原酶NIA3蛋白基因OsNia3及其应用 Download PDFInfo
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Abstract
水稻硝酸还原酶NIA3蛋白基因OsNia3，其cDNA序列如SEQ ID NO.1所示，其编码的水稻NIA3蛋白，氨基酸序列如SEQ ID NO.2所示；利用转基因技术获得水稻基因OsNia3的纯合敲除突变体和纯合OsNia3过表达株系，分析发现OsNia3基因敲除株系的株高变矮，生育期缩短，单株产量减少；而OsNia3过表达株系的株高较高，生育期延长，单株产量增加，水稻硝酸还原酶NIA3蛋白基因OsNia3可作为目的基因导入植物，以提高植物氮素利用效率，进行植物品种改良。
Description
技术领域
本发明涉及基因工程技术领域，尤其涉及一种水稻硝酸还原酶NIA3蛋白基因OsNia3及其应用。
背景技术
植物主要以硝酸盐和铵盐的形式从土壤中吸收氮元素，硝酸盐同化是受到高度调控的氮素利用过程[1]。硝酸盐降解的第一步发生在细胞质中，由硝酸还原酶(Nitratereductase,NR)将硝酸盐还原成亚硝酸盐，亚硝酸盐进入叶绿体或质体后，经过后续一系列的代谢反应被植物体所利用。目前研究发现，拟南芥中存在两个硝酸还原酶基因(AtNia1和AtNia2)，其编码分别是NIA1和NIA2蛋白[2]。敲除AtNia1和AtNia2后，拟南芥突变体NR活性显著降低，而nia1nia2双敲除突变体中几乎检测不到NR活性[3]，在拟南芥中过量表达Nia基因，能够增强转基因株系的NR活性，降低植株内硝酸盐的积累[4]。拟南芥中NR活性主要受基因AtNia2控制，AtNia2基因突变将导致90％NR活性的丧失[5]。水稻中含有2个硝酸还原酶基因，分别为OsNia1和OsNia2[6]。从NCBI中(http://www.ncbi.nlm.nih.gov/)搜索OsNia1基因mRNA序列过程中，发现OsNia1基因的mRNA与[NADH]1-like(LOC4345798)的mRNA的同源性为99％，后续实验将其命名为OsNia3。研究证明，敲除和过表达OsNia3基因能够降低和升高水稻叶片中的NR活性，提高氮素利用效率，对水稻的生长发育也有一定的影响。
上述涉及的参考文献如下：
[1]Crawford N M.Nitrate:nutrient and signal for plant growth[J].PlantCell,1995,7(7):859-868；
[2]Choi H K,Kleinhofs A,An G.Nucleotide sequence of rice nitratereductase genes[J].Plant Molecular Biology,1989(13):731-733；
[3]Wilkinson J Q,Crawford N M.Identification and characterization ofa chlorate-resistant mutant of Arabidopsis thaliana with mutations in bothnitrate reductase structural genes NIA1 and NIA2[J].Molecular&generalgenetics:MGG,1993,239(1-2):289；
[4]Curtis I S,Power J B,de Laat A M M,et al.Expression of a chimericnitrate reductase gene in transgenic lettuce reduces nitrate in leaves[J].Plant Cell Reports,1999,18(11):889-896；
[5]Yu X,Sukumaran S,Márton L.Differential Expression of theArabidopsis Nia1 and Nia2 Genes1:Cytokinin-Induced Nitrate Reductase ActivityIs Correlated With Increased[J].Plant Physiology,1998,116(3):1091-1096；
[6]Hasegawa H,Katagiri T,Ida S,et al.Characterization of a rice(Oryzasativa L.)mutant deficient[J].Theor Appl Genet,1992(84):6-9。
发明内容
本发明所解决的技术问题在于提供一种水稻硝酸还原酶NIA3蛋白基因OsNia3及其应用，以解决上述背景技术中的问题。
本发明所解决的技术问题采用以下技术方案来实现：
水稻硝酸还原酶NIA3蛋白基因OsNia3，其cDNA序列如SEQ ID NO.1所示。
利用水稻硝酸还原酶NIA3蛋白基因OsNia3编码的水稻蛋白NIA3，其氨基酸序列如SEQ ID NO.2所示。
将水稻硝酸还原酶NIA3蛋白基因OsNia3应用在水稻转基因植株上，具体步骤如下：
1)提取总RNA
选用水稻品种Kitaake作为RNA的提取材料，待水稻幼苗生长至约20d，取叶片液氮冻存；而后取部分液氮冻存叶片，用研钵研碎，按照RNAprep Pure植物总RNA提取试剂盒说明提取水稻叶片总RNA，再利用NanoDrop ND2000超微量核酸蛋白分析仪对其浓度和质量进行检测，利用1％琼脂糖凝胶电泳检测其完整性；
2)克隆水稻基因OsNia3
从NCBI中(http://www.ncbi.nlm.nih.gov/)搜索Nia1基因的mRNA序列，发现Nia1基因的mRNA与[NADH]1-like(LOC4345798)的mRNA高度同源(同源性99％)，后续实验将其命名为OsNia3，根据水稻OsNia3的全长序列，设计两端引物P1:SEQ ID NO.3和P2:SEQ IDNO.4；
SEQ ID NO.3：5-TGAACGCAGAACCGAACAC-3；
SEQ ID NO.4：5-TCCACGGGCCACCATAC-3；
将步骤1)获得的水稻叶片总RNA转录合成cDNA第一链，并以cDNA第一链为模版，采用高保真KOD FX酶进行PCR扩增，利用琼脂糖凝胶回收试剂盒进行回收，将目的基因与pCUbi1390-GFP过表达载体进行连接获得过表达载体pCUbi1390-GFP-Nia3，测序获得水稻基因OsNia3的cDNA序列SEQ ID NO.1及其编码的水稻蛋白NIA3氨基酸序列SEQ ID NO.2；
3)构建水稻基因OsNia3敲除载体pCas9-OsNia3
根据步骤2)获得的水稻基因OsNia3的全长cDNA序列，利用CRISPR-P在线网站(http://cbi.hzau.edu.cn/cgi-bin/CRISPR)设计引物片段，而后将设计的引物片段序列与pCas9载体上的sgRNA骨架部分序列一起黏贴至RNA fold Web server中进行RNA结构预测，获得适合的引物片段P3:SEQ ID NO.5，再将引物片段P3:SEQ ID NO.5加上接头序列获得引物片段P4:SEQ ID NO.6及其反向互补序列P5:SEQ ID NO.7，交由公司合成后，将引物片段P4与反向互补序列P5利用PCR仪完成退火结合以获得退火产物，最后将退火产物(即水稻基因OsNia3的18bp序列)克隆至pCas9载体，测序鉴定确保目的基因序列正确，得水稻基因OsNia3敲除载体pCas9-OsNia3；
SEQ ID NO.5：5-GCTCGGGGAACCGCCGCA-3；
SEQ ID NO.6：5-GCTCGGGGAACCGCCGCAGTTTTAGAGCTATGCTGAAAAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3；
SEQ ID NO.7：
5-GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTTTTCAGCATAGCTCTAAAACTGCGGCGGTTCCCCGAGC-3；
4)构建水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3
根据水稻基因OsNia3的cDNA序列SEQ ID NO.1，设计扩增处完整编码的携带限制性酶切位点Hand III和BamH I的上游引物P6:SEQ ID NO.8和下游引物P7:SEQ ID NO.9；
SEQ ID NO.8：5-TCTGCACTAGGTACCTGCAGATGGCTGCTTCCGTGC-3；
SEQ ID NO.9：5-ATGGATCCGTCGACCTGCAGGAACACGATGAAAGAATTGGCC-3；
以步骤2)获得的过表达载体pCUbi1390-GFP-Nia3为模版，经PCR扩增后，将水稻基因OsNia3的cDNA克隆至载体双元表达载体pCUBi1390-GFP中Hand III和BamH I酶切位点，测序鉴定确保表达载体中编码区阅读框架正确，得水稻基因OsNia3过表达载体pCUBi1390-GFP-OsNia3；
5)获得纯合OsNia3过表达株系
将步骤3)获得的水稻基因OsNia3敲除载体pCas9-OsNia3转入农杆菌，进一步转入粳稻品种Kitaake中，转基因植株经PCR和测序鉴定，获得OsNia3基因突变且无载体骨架的纯合敲除突变体nia3，水稻突变体nia3表现为NR活性降低，硝态氮的转化速率降低，株高降低，生育期缩短，单株产量减少；
将步骤4)获得的水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3转入农杆菌，进一步转入纯合敲除突变体nia3中，转基因植株经PCR和RT-PCR验证获得纯合OsNia3过表达株系，OsNia3过表达株系表现为NR活性升高，硝态氮的转化速率升高，株高升高，生育期延长。
有益效果：
1)本发明公开水稻硝酸还原酶NIA3蛋白基因OsNia3及其所编码的蛋白质，水稻硝酸还原酶NIA3蛋白基因OsNia3为水稻中首次报道，转基因实验证明该基因的敲除和过量表达可降低和提高水稻的NR活性、氮素利用效率，因此有望作为目的基因导入植物，以提高植物氮素利用效率，进行植物品种改良；
2)本发明对获得转基因株系进行PCR分子鉴定及测序验证后，获得所述的水稻基因OsNia3的纯合敲除株系和过表达株系，对转基因水稻的生长发育进行分析，发现OsNia3基因敲除株系的株高变矮，生育期缩短，单株产量减少；而OsNia3过表达株系的株高较高，生育期延长，单株产量增加。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。
将水稻硝酸还原酶NIA3蛋白基因OsNia3应用在水稻转基因植株上，具体步骤如下：
1)总RNA提取
选用水稻品种Kitaake作为RNA的提取材料，待水稻幼苗生长至约20d，取叶片液氮冻存；而后取部分液氮冻存叶片，用研钵研碎，按照RNAprep Pure植物总RNA提取试剂盒说明提取水稻叶片总RNA，再利用NanoDrop ND2000超微量核酸蛋白分析仪对其浓度和质量进行检测，利用1％琼脂糖凝胶电泳检测其完整性；
2)水稻基因OsNia3的克隆
从NCBI中(http://www.ncbi.nlm.nih.gov/)搜索Nia1基因的mRNA序列，发现Nia1基因的mRNA与[NADH]1-like(LOC4345798)的mRNA高度同源(同源性99％)，后续实验将其命名为OsNia3，根据水稻OsNia3的全长序列，设计两端引物P1:SEQ ID NO.3和P2:SEQ IDNO.4；
SEQ ID NO.3：5-TGAACGCAGAACCGAACAC-3；
SEQ ID NO.4：5-TCCACGGGCCACCATAC-3；
将步骤1)获得的水稻叶片总RNA转录合成cDNA第一链，并以cDNA第一链为模版，采用高保真KOD FX酶进行PCR扩增，利用琼脂糖凝胶回收试剂盒进行回收，将目的基因与pCUbi1390-GFP过表达载体进行连接获得过表达载体pCUbi1390-GFP-Nia3，测序获得水稻基因OsNia3的cDNA序列SEQ ID NO.1及其编码的水稻蛋白NIA3氨基酸序列SEQ ID NO.2；
3)水稻基因OsNia3敲除载体pCas9-OsNia3的构建
根据步骤2)获得的水稻基因OsNia3的全长cDNA序列，利用CRISPR-P在线网站(http://cbi.hzau.edu.cn/cgi-bin/CRISPR)设计引物片段，而后将设计的引物片段序列与pCas9载体上的sgRNA骨架部分序列一起黏贴至RNA fold Web server中进行RNA结构预测，获得适合的引物片段P3:SEQ ID NO.5，再将引物片段P3:SEQ ID NO.5加上接头序列获得引物片段P4:SEQ ID NO.6及其反向互补序列P5:SEQ ID NO.7，交由公司合成后，将引物片段P4与反向互补序列P5利用PCR仪完成退火结合以获得退火产物，最后将退火产物(即水稻基因OsNia3的18bp序列)克隆至pCas9载体，测序鉴定确保目的基因序列正确，得水稻基因OsNia3敲除载体pCas9-OsNia3；
SEQ ID NO.5：5-GCTCGGGGAACCGCCGCA-3；
SEQ ID NO.6：5-GCTCGGGGAACCGCCGCAGTTTTAGAGCTATGCTGAAAAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3；
SEQ ID NO.7：5-GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTTTTCAGCATAGCTCTAAAACTGCGGCGGTTCCCCGAGC-3；
4)水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3的构建
根据水稻基因OsNia3的cDNA序列SEQ ID NO.1，设计扩增处完整编码的携带限制性酶切位点Hand III和BamH I的上游引物P6:SEQ ID NO.8和下游引物P7:SEQ ID NO.9；
SEQ ID NO.8：5-TCTGCACTAGGTACCTGCAGATGGCTGCTTCCGTGC-3；
SEQ ID NO.9：5-ATGGATCCGTCGACCTGCAGGAACACGATGAAAGAATTGGCC-3；
以步骤2)中获得的过表达载体pCUbi1390-GFP-Nia3为模版，经PCR扩增后，将水稻基因OsNia3的cDNA克隆至载体双元表达载体pCUBi1390-GFP中Hand III和BamH I酶切位点，测序鉴定确保表达载体中编码区阅读框架正确，得水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3；
5)纯合OsNia3过表达株系的获得
将步骤3)获得的水稻基因OsNia3敲除载体pCas9-OsNia3转入农杆菌，进一步转入粳稻品种Kitaake中，转基因植株经PCR和测序鉴定，获得OsNia3基因突变且无载体骨架的纯合敲除突变体nia3，水稻突变体nia3表现为NR活性降低，硝态氮的转化速率降低，株高降低，生育期缩短，单株产量减少；
将步骤4)获得的水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3转入农杆菌，进一步转入纯合敲除突变体nia3中，转基因植株经PCR和RT-PCR验证获得纯合OsNia3过表达株系，OsNia3过表达株系表现为NR活性升高，硝态氮的转化速率升高，株高升高，生育期延长。
序列表
<110> 江西农业大学
<120> 一种水稻硝酸还原酶NIA3蛋白基因OsNia3及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3315
<212> DNA
<213> 水稻基因OsNia3的cDNA序列()
<400> 1
gcgcctcttc caggccacta cttctctggg ctctggctac tcttcgacgg cggccggcgt 60
agcgagctgt agctgagctg aacgcagaac cgaacaccaa gcatggccgc ttccgtgcag 120
ccgcggcagt tcggccacct cgagccgggc tccgcgccgg tgtgcggcgc cgcatcctcg 180
aacggcgcca aggcgtaccc tcccgcgaac ggcatcccgc gccgcgccga ctcaccggtg 240
cgcgggtgcg gcttccctcc cctcgtctcg ccaccttcgc ggaagccgcc cagcgatggg 300
tcggacgacg aggaggagga gcaggaggac tggcgggagc tgtacggctc gcacctgcag 360
ctggaggtgg aaccgtcggt gcgcgacgcg cgcgacgagg gcaccgccga cgcgtggatc 420
gagcgcaacc cgttgctgat ccggctcacc gggaaacacc cgctgaactg cgaggcgccg 480
ctggcgaggc tcatgcacca cggcttcatc accccggctg cgctgcactt cgtgcgcaac 540
cacggcgcag tgccgcgggg tgactggtcg acgtggaccg tcgaggtgac ggggctcgtc 600
aagcgtccca tgcggctcac cgtggacgag ctggtcaacg gcttccccgc cgtggaggtc 660
cccgtcacgc tggcctgctc ggggaaccgc cgcaaggagc agaacatggt gcagcagact 720
gtggggttca actttggcgc cgccgccgtg tccacgtcgg tgtggcacgg cgcccgcctc 780
cgcgacgtgc tccggcggtg cggcatcatg cccagcaagg gcggtgcgct caacgtgtgc 840
ttcgagggcg ccgaggacct ccccggcggc ggcggctcca agtacggcac cagcatcaca 900
cgccagtggg cgctggaccc gtcgcgggac atcatgctcg cctacatgca gaatggcgag 960
ccgctgctcc ccgaccacgg cttccccgtc cgcgccatca tccccggctg caccggcggc 1020
cgcatggtca agtgggtcaa gcgcatcatc gtcaccaccg ccgagtccga caactactac 1080
cattacaagg acaaccgcgt cttcccgtcc catgtcgacg ccgagctcgc caacgccgat 1140
gcgtggtggt acaagccgga gtacatcatc aacgagctga acgtgaactc ggtgatcacg 1200
gcgcccgggc acgacgagat cctgcccatc aacggcatca ccacgcagcg cggctacacc 1260
atgaagggat acgcctactc cggcggcggc aagaggatca cgcgggtgga ggtgacgctg 1320
gacggcggcg agacatggct ggtgtgcgtg ctggacctcc cggagaagcc caccaagtac 1380
ggcaagcact ggtgctggtg cttctggtcc gtcgaggtcg aggtgctcga cctcctcggc 1440
gccaaggaga tcgccgtgcg cgcctgggac cagtcgcaca acacccagcc cgagaagctc 1500
atctggaatc tcatggggat gatgaacaac tgctggttca aggtgaaggt gaacgtgtgc 1560
cggccgcaca agggtgagat cgggctggtg ttcgagcacc cgacgcagcc cgggaaccag 1620
accggcgggt ggatggcgag gcagaagcac ctggagacgg cggaggcggc cgcaccgggg 1680
ctgaagcgga gcacgtcgac gccgttcatg aacaccaccg acggcaagca gtttaccatg 1740
tccgaggtgc gcaagcactc gtcgcaggac tcggcgtgga tcgtcgtcca cggtcacgtc 1800
tacgactgca cggccttcct caaggaccac cccggcggcg ccgacagcat cctcatcaac 1860
gccggcaccg actgcaccga ggagttcgac gccatccact ccgacaaggc caaggcgctc 1920
ctcgacacct accgcatcgg cgagctcatc accaccggcg ccgggtacag ctccgacaac 1980
tccgtccacg gcgcgtccaa cctctcccag ctcgccccca tccgcgaggc catcaaggcg 2040
ccggcgcccg tcgcgctctc cagcccgcgc gacaaggtcc cctgccaact cgtcgacaag 2100
aaggagctct cccgcgacgt ccgcctcttc cgcttcgcgc tgccgtcctc cgaccaggtg 2160
ctcggcctcc ccgtcggcaa gcacatcttc gtgtgcgcca gcatcgaagg gaagctgtgc 2220
atgcgggcgt acacgccgac gagcatggtc gacgaggtcg gccacttcga cctcctcatc 2280
aaggtgtact tcaagaacga gcaccccaag ttccccgatg gcgggctcat gacgcagtac 2340
ctggactcgc tccccgtggg cgcctacatc gacgtcaagg ggccactcgg ccacgtcgag 2400
tacaccggcc gcggcgagtt cgtcatcaac ggcaagccgc ggaacgcgcg gcggctggcg 2460
atgatcgccg gcgggagcgg gatcacgccc atgtaccagg tcatccagtc ggtgctgcgc 2520
gaccagccgg aggacacgac ggagatgcac ctggtgtacg cgaaccggac ggaggacgac 2580
atcctcctcc gcgacgagct cgaccggtgg gcggcggagt acccggacag gctcaaggtg 2640
tggtacgtca tcgaccaggt gaagcggccg gaggaagggt ggaagtacgg cgtcgggttc 2700
gtcacggagg aggtgctgcg ggagcacgtg ccggagggcg gcgacgacac gctcgcgctc 2760
gcctgcgggc cgccgccgat gatcaagttc gccgtctcgc cgaacctgga gaagatgaag 2820
tacgacatgg ccaattcttt catcgtgttc taaactccta ctactaaatt atacgcaccg 2880
cattaattat tttggtatac gtacgacgat gtatatacgt gtcgtgccaa ttagaaacga 2940
aatgctgcat gcatgcgtat gcaagatgag agaaaaaaaa acaatatcta agttgtagta 3000
gtgtactctt gataatatcc tagagtacat actgcataca taggccatga tttccctttc 3060
taagaggcta gcggttgatt ccttcctgta tactatgtta cgaaccaagg ggggattggg 3120
aattttggat tgaacgagca gaggaagaat agaaacagag gaagaactcg gaagaacaga 3180
gtagtttggg agagaagcag taggattatt cagagatagt gttttgatta caaactagta 3240
tggtggcccg tggagattgc gcggctagca tcattatatt ttctctcata taattacata 3300
tatgatttct catta 3315
<210> 2
<211> 629
<212> DNA
<213> 水稻硝酸还原酶NIA3蛋白基因OsNia3编码的水稻蛋白NIA3氨基酸序列()
<220>
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<222> (19)..(19)
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<400> 2
maasvrghgs avcgaassng akayangrra dsvrgcgvss rksdgsdddw rygshvsvrd 60
ardgtadawr nrtgkhncaa rmhhgtaahv rnhgavrgdw stwtvvtgvk rmrtvdvnga 120
vvvtacsgnr rknmvtvgng aaavstsvwh garrdvrrcg mskgganvcg adggggskyg 180
tstrwadsrd maymngdhgv ragctggrmv kwvkrvttas dnyyhykdnr vshvdaanad 240
awwykynnvn svtaghdngt trgytmkgya ysgggkrtrv vtdggtwvcv dktkygkhwc 300
wcwsvvvdga kavrawdshn tkwnmgmmnn cwkvkvnvcr hkggvhtgnt ggwmarkhta 360
aaagkrstst mnttdgktms vrkhssdsaw vvhghvydct akdhggadsn agtdctdahs 420
dkakadtyrg ttgagyssdn svhgasnsar akaavassrd kvcvdkksrd vrrassdvgv 480
gkhvcasgkc mrayttsmvd vghdkvyknh kdggmtydsv gaydvkgghv ytgrgvngkr 540
narramaggs gtmyvsvrdd ttmhvyanrt ddrddrwaay drkvwyvdvk rgwkygvgvt 600
vrhvggddta acgmkavsnk mkydmansv 629
Claims (10)
1.水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，其cDNA序列如SEQ ID NO.1所示。
2.根据权利要求1所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，利用水稻硝酸还原酶NIA3蛋白基因OsNia3编码的水稻蛋白NIA3，其氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，应用于水稻基因OsNia3敲除载体pCas9-OsNia3。
4.根据权利要求3所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，水稻基因OsNia3敲除载体pCas9-OsNia3是将是将水稻硝酸还原酶NIA3蛋白基因OsNia3的18bp序列连接至pCas9载体所得。
5.根据权利要求1所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，应用于水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3。
6.根据权利要求5所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3是将水稻基因OsNia3的cDNA克隆至载体双元表达载体pCUBi1390-GFP中Hand III和BamH I酶切位点所得。
7.根据权利要求1所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，应用在水稻转基因植株上，具体步骤如下：
1)提取总RNA
选用水稻品种Kitaake作为RNA的提取材料，待水稻幼苗生长至约20d，取叶片液氮冻存；而后取部分液氮冻存叶片，用研钵研碎，按照RNAprep Pure植物总RNA提取试剂盒说明提取水稻叶片总RNA；
2)克隆水稻基因OsNia3
从NCBI中搜索Nia1基因的mRNA序列，发现Nia1基因的mRNA与[NADH]1-like的mRNA高度同源，后续实验将其命名为水稻基因OsNia3，根据水稻基因OsNia3的全长序列，设计两端引物P1:SEQ ID NO.3和P2:SEQ ID NO.4；
将步骤1)获得的水稻叶片总RNA转录合成cDNA第一链，并以cDNA第一链为模版，采用高保真KOD FX酶进行PCR扩增，利用琼脂糖凝胶回收试剂盒进行回收，将目的基因与pCUbi1390-GFP过表达载体进行连接获得过表达载体pCUbi1390-GFP-Nia3，测序获得水稻基因OsNia3的cDNA序列SEQ ID NO.1及其编码的水稻蛋白NIA3氨基酸序列SEQ ID NO.2；
3)构建水稻基因OsNia3敲除载体pCas9-OsNia3
根据步骤2)获得的水稻基因OsNia3的全长cDNA序列，利用CRISPR-P在线网站设计引物片段，而后将设计的引物片段序列与pCas9载体上的sgRNA骨架部分序列一起黏贴至RNAfold Web server中进行RNA结构预测，获得引物片段P3:SEQ ID NO.5，再将引物片段P3:SEQ ID NO.5加上接头序列获得引物片段P4:SEQ ID NO.6及其反向互补序列P5:SEQ IDNO.7，交由公司合成后，将引物片段P4与反向互补序列P5的引物利用PCR仪完成退火结合以获得退火产物，最后将退火产物克隆至pCas9载体，测序鉴定确保目的基因序列正确，得水稻基因OsNia3敲除载体pCas9-OsNia3；
4)构建水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3
根据水稻基因OsNia3的cDNA序列SEQ ID NO.1，设计扩增处完整编码的携带限制性酶切位点Hand III和BamH I的上游引物P6:SEQ ID NO.8和下游引物P7:SEQ ID NO.9；
以步骤2)中获得的过表达载体pCUbi1390-GFP-Nia3为模版，经PCR扩增后，将水稻基因OsNia3的cDNA克隆至载体双元表达载体pCUBi1390-GFP中Hand III和BamH I酶切位点，测序鉴定确保表达载体中编码区阅读框架正确，得水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3；
5)获得纯合OsNia3过表达株系
将步骤3)获得的水稻基因OsNia3敲除载体pCas9-OsNia3转入农杆菌，进一步转入粳稻品种Kitaake中，转基因植株经PCR和测序鉴定，获得OsNia3基因突变且无载体骨架的纯合敲除突变体nia3；
将步骤4)获得的水稻基因OsNia3表达载体pCUBi1390-GFP-OsNia3转入农杆菌，进一步转入纯合敲除突变体nia3中，转基因植株经PCR和RT-PCR验证获得纯合OsNia3过表达株系。
8.根据权利要求7所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，步骤2)中，两端引物具体序列如下：
SEQ ID NO.3：5-TGAACGCAGAACCGAACAC-3；
SEQ ID NO.4：5-TCCACGGGCCACCATAC-3。
9.根据权利要求7所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，步骤3)中，引物具体序列如下：
SEQ ID NO.5：5-GCTCGGGGAACCGCCGCA-3；
SEQ ID NO.6：5-GCTCGGGGAACCGCCGCAGTTTTAGAGCTATGCTGAAAAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3；
SEQ ID NO.7：5-GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTTTTCAGCATAGCTCTAAAACTGCGGCGGTTCCCCGAGC-3。
10.根据权利要求7所述的水稻硝酸还原酶NIA3蛋白基因OsNia3，其特征在于，步骤4)中，反向突变引物P5:SEQ ID NO.6具体序列如下：
SEQ ID NO.8：5-TCTGCACTAGGTACCTGCAGATGGCTGCTTCCGTGC-3；
SEQ ID NO.9：5-ATGGATCCGTCGACCTGCAGGAACACGATGAAAGAATTGGCC-3。
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| CN112342220A (zh) * | 2020-11-30 | 2021-02-09 | 湖南农业大学 | 水稻糖转运基因OsMST1及其糖转运体、应用和扩增引物 |
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| CN102260591A (zh) * | 2011-07-01 | 2011-11-30 | 华南农业大学 | 一种食用油中苯并芘的去除方法 |
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