一个CRISPR/Cas9-VQR基因编辑系统的构建
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摘要
CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术,但该编辑系统的使用范围受PAM (proto-spacer-motif)位点NGG的限制。本研究通过突变Streptococcus pyogenes Cas9 (SpCas9)编码氨基酸(1135位的天冬氨酸D突变成缬氨酸V, 1335位的精氨酸R突变为谷胱氨酸Q, 1337位的苏氨酸T突变为精氨酸R,命名该突变子为Cas9-VQR)改造其识别PAM为NGA的位点以扩大其使用范围。并使用玉米Ubi启动子启动Cas9-VQR基因、优化SpCas9的密码子、加入保守的核定位信号序列、增加单子叶植物中保守的3′UTR序列和使用水稻U6启动子启动gRNA来修饰该编辑系统。结果表明Cas9-VQR系统能够识别PAM为NGA的位点,并进行有效的切割。体外酶切活性检测结果表明Cas9-VQR的切割效率为5%～70%。水稻转化检测结果表明Cas9-VQR的切割效率约为27.5%～70.5%,平均切割效率为46.23%。本研究拓宽了CRISPR/Cas9系统在作物中的使用范围,特别是NGA PAM位点较高的作物。
Clustered Regularly Interspaced Short Palindromic Repeat and Cas9(CRISPR/Cas9), a new generation of genomeediting technology, is widely applied among bacteria, yeast, animals and plants, however, the typical CRISRP/Cas9 cannot recognize the NGA proto-spacer-motif(PAM), which limits its application. In order to broaden the applications of CRIPSR/Cas9 system, we modified the Streptococcus pyogenes Cas9(SpCas9) sequence by the PCR site-direct mutagenesis, which encodes V(1135), Q(1335), and R(1337), to make the CRIPSR/Cas9-VQR able to recognize the NGA PAM motif. We also constructed a binary expression vector of CRISRP/Cas9-VQR with maize ubiquitin as the promoter to drive the Cas9-VQR, optimizing SpCas9-codon, adding conserved nuclear localization signal sequence, and increasing the conserved 3' UTR sequence of monocots, and using OsU6 transcripts of sRNA. CRISPR/Cas9-VQR could recognize the NGA motif and cut targeted sequence in vivo. We assembled the Cas9-VQR protein with the s RNAs in vitro. The Cas9-VQR could cleave the targeted fragments with about 5%–70% of mutation efficiency. In the transformation of rice, we detected about 27.50%–70.50% of mutation ratio, with an average of 46.23%. This system broadens the CRISPR/Cas9 applications in crops, especially in these with higher PAM locus of NGA.
Clustered Regularly Interspaced Short Palindromic Repeat and Cas9(CRISPR/Cas9), a new generation of genomeediting technology, is widely applied among bacteria, yeast, animals and plants, however, the typical CRISRP/Cas9 cannot recognize the NGA proto-spacer-motif(PAM), which limits its application. In order to broaden the applications of CRIPSR/Cas9 system, we modified the Streptococcus pyogenes Cas9(SpCas9) sequence by the PCR site-direct mutagenesis, which encodes V(1135), Q(1335), and R(1337), to make the CRIPSR/Cas9-VQR able to recognize the NGA PAM motif. We also constructed a binary expression vector of CRISRP/Cas9-VQR with maize ubiquitin as the promoter to drive the Cas9-VQR, optimizing SpCas9-codon, adding conserved nuclear localization signal sequence, and increasing the conserved 3' UTR sequence of monocots, and using OsU6 transcripts of sRNA. CRISPR/Cas9-VQR could recognize the NGA motif and cut targeted sequence in vivo. We assembled the Cas9-VQR protein with the s RNAs in vitro. The Cas9-VQR could cleave the targeted fragments with about 5%–70% of mutation efficiency. In the transformation of rice, we detected about 27.50%–70.50% of mutation ratio, with an average of 46.23%. This system broadens the CRISPR/Cas9 applications in crops, especially in these with higher PAM locus of NGA.
引文
[1]Song G Y,Jia M L,Chen K,Kong X C,Khattak B,Xie C X,Li AL,Mao L.CRISPR/Cas9:A powerful tool for crop genome editing.Crop J,2016,4:75-82.
[2]Shukla V K,Doyon Y,Miller J C,DeKelver R C,Moehle E A,Worden S E,Mitchell J C,Arnold N L,Gopalan S,Meng X,Choi V M,Rock J M,Wu Y Y,Katibah G E,Zhifang G,McCaskill D,Simpson M A,Blakeslee B,Greenwalt S A,Butler H J,Hinkley SJ,Zhang L,Rebar E J,Gregory P D,Urnov F D.Precise genome modification in the crop species Zea mays using zinc-finger nucleases.Nature,2009,459:437-441.
[3]Schiml S,Fauser F,Puchta H.The CRISPR/Cas9 system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny.Plant J,2014,80:1139-1150.
[4]Shukla V K,Doyon Y,Miller J C,DeKelver R C,Moehle E A,Worden S E,Mitchell J C,Arnold N L,Gopalan S,Meng X,Choi V M,Rock J M,Wu Y Y,Katibah G E,Zhifang G,McCaskill D,Simpson M A,Blakeslee B,Greenwalt S A,Butler H J,Hinkley SJ,Zhang L,Rebar E J,Gregory P D,Urnov F D.Precise genome modification in the crop species Zea mays using zinc-finger nucleases.Nature,2009,459:437-411.
[5]Fauser F,Schiml S,Puchta H.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.Plant J,2014,2:348-359.
[6]Li J F,Norville J E,Aach J,McCormack M,Zhang D,Bush J,Church G M,Sheen J.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nat Biotechnol,2013,31:688-691.
[7]Fauser F,Schiml S,Puchta H.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.Plant J,2014,79:348-359.
[8]Feng Z,Zhang B,Ding W,Liu X,Yang DL,Wei P,Cao F,Zhu S,Zhang F,Mao Y,Zhu J K.Efficient genome editing in plants using a CSRIPR/Cas system.Cell Res,2013,23:1229-1232.
[9]Hyun Y B,Kim J,Cho S W,Choi Y,Kim J S,Coupland G.Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted REGN of the CRISPR/Cas9 system to generate heritable null alleles.Planta,2015,241:271-284.
[10]Feng Z,Mao Y,Xu N,Zhang B,Wei P,Yang D L,Wang Z,Zhang Z,Zheng R,Yang L,Zeng L,Liu X,Zhu J K.Multigeneration analysis reveals the inheritance,specificity,and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.Proc Natl Acad Sci USA,2014,111:4632-4637.
[11]Ali Z,Abul-faraj A,Li L,Ghosh N,Piatek M,Mahjoub A,Aouida M,Piatek A,Baltes N J,Voytas D F,Dinesh-Kumar S,Mahfouz M M.Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system.Mol Plant,2015,8:1288-1291.
[12]Gao J,Wang G,Ma S,Xie X,Wu X,Zhang X,Wu Y,Zhao P,Xia Q.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.Plant Mol Biol,2015,87:99-110.
[13]Nekrasov V,Staskawicz B,Weigel D,Jones J D,Kamoun S.Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.Nat Biotechnol,2013,31:691-693.
[14]Shan Q,Wang Y,Li J,Zhang Y,Chen K,Liang Z,Zhang K,Liu J,Xi J J,Qiu J L,Gao C.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol,2013,31:686-688.
[15]Miao J,Guo D S,Zhang J Z,Huang Q P,Qin G J,Zhang X,Wan J M,Gu H Y,Qu L J.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Res,2013,23:1233-1236.
[16]Zhang H,Zhang J,Wei P,Zhang B,Gou F,Feng Z,Mao Y,Yang L,Zhang H,Xu N,Zhu J K.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.Plant Biotechnol J,2014,12:797-807.
[17]Xie K B,Yang Y N.RNA-guided genome editing in plants using a CRISPR-Cas system.Mol Plant,2013,6:1975-1983.
[18]Xu R,Li H,Qin R,Wang L,Li L,Wei P,Yang J.Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.Rice,2014,1:5-7.
[19]Xie K B,Minkenberg B,Yang Y N.Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.Proc Natl Acad Sci USA,2015,112:3570-3575.
[20]Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,Zhao X,Dong Z,Liu Y G.A robust CRISPR/Cas9 system for convenient high-efficiency multiplex genome editing in monocot and dicot plants.Mol Plant,2015,8:1274-1284.
[21]Zhou H,Liu B,Weeks D P,Spalding M H,Yang B.Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice.Nucleic Acids Res,2014,42:10903-10914.
[22]Sun X,Hu Z,Chen R,Jiang Q,Song G,Zhang H,Xi Y.Targeted mutagenesis in soybean using the CRISPR-Cas9 system.Sci Rep,2015,5:10342.
[23]Pan C T,Ye L,Qin L,Liu X,He Y J,Wang J,Chen L F,Lu G.CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations.Sci Rep,2016,6:24765.
[24]Jiang W,Zhou H,Bi H,Fromm M,Yang B,Weeks D P.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice.Nucleic Acids Res,2013,41:e188.
[25]Liang Z,Zhang K,Chen K,Gao C.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.J Genet Genomics,2014,41:63-68.
[26]Li C,Unver T,Zhang B H.A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton(Gossypium hirsutum L.).Sci Rep,2017,7:43902.
[27]Shan Q W,Wang Y P,Li J,Gao C X.Genome editing in rice and wheat using the CRISPR/Cas9 system.Nat Protocol,2014,9:2395-2410.
[28]Li B,Cui G,Shen G,Zhan Z,Huang L,Chen J,Qi X.Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza.Sci Rep,2017,7:43320.
[29]Xu R,Li H,Qin R,Wang L,Li L,Wei P,Yang J.Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.Rice,2014,7:1-5.
[30]Mao Y,Zhang H,Xu N,Zhang B,Gou F,Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants.Mol Plant,2013,6:2008-2011
[31]Xing H L,Dong L,Wang Z P,Zhang H Y,Han C Y,Liu B,Wang X C,Chen Q J.A CRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol,2014,14:327-328.
[32]Li J F,Norville J E,Aach J,McCormack M,Zhang D,Bush J,Church G M,Sheen J.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nat Biotechnol,2013,8:688.
[33]Liu W,Zhu X,Lei M,Xia Q,Botella J R,Zhu J K,Mao Y.A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana.Sci Bull,2015,15:1332-1347.
[34]Wang Y,Cheng X,Shan Q,Zhang Y,Liu J,Gao C,Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol,2014,32:947-951.
[35]Wang F,Wang C,Liu P,Lei C,Hao W,Gao Y,Liu Y G,Zhao K.Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922.PLoSOne,2016,11:e0154027.
[36]Ran F A,Cong L,Yan W X,Scott D A,Gootenberg J S,Kriz A J,Zetsche B,Shalem O,Wu X,Makarova K S,Koonin E V,Sharp P A,Zhang F.In vivo genome editing using Staphylococcus aureus Cas9.Nature,2015,520:186-191.
[37]Kleinstiver B P,Prew M S,Tsai S Q,Topkar V V,Nguyen N T,Zheng Z,Gonzales A P,Li Z,Peterson R T,Yeh J R,Aryee M J,Joung J K.Engineering CRISPR-Cas9 nucleases with altered PAM specificities.Nature,2015,523:481-485.
[38]Hu X X,Wang C,Fu Y P,Liu Q,Jiao X Z,Wang K J.Expanding the range of CRISPR/Cas9 genome editing in rice.Mol Plant,2016,9:943-945.
[39]Hu X X,Meng X B,Liu Q,Li J Y,Wang C J.Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.Plant Biotechnol J,2018,16:292-297.
[40]Hiei Y,Komari T,Kubo T.Transformation of rice mediated by Agrobacterium tumefaciens.Plant Mol Biol,1997,35:205-218.
[41]Wang K,Liu H Y,Du L P,Ye X G.Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.Plant Biotechnol J,2017,15:614-623.
[2]Shukla V K,Doyon Y,Miller J C,DeKelver R C,Moehle E A,Worden S E,Mitchell J C,Arnold N L,Gopalan S,Meng X,Choi V M,Rock J M,Wu Y Y,Katibah G E,Zhifang G,McCaskill D,Simpson M A,Blakeslee B,Greenwalt S A,Butler H J,Hinkley SJ,Zhang L,Rebar E J,Gregory P D,Urnov F D.Precise genome modification in the crop species Zea mays using zinc-finger nucleases.Nature,2009,459:437-441.
[3]Schiml S,Fauser F,Puchta H.The CRISPR/Cas9 system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny.Plant J,2014,80:1139-1150.
[4]Shukla V K,Doyon Y,Miller J C,DeKelver R C,Moehle E A,Worden S E,Mitchell J C,Arnold N L,Gopalan S,Meng X,Choi V M,Rock J M,Wu Y Y,Katibah G E,Zhifang G,McCaskill D,Simpson M A,Blakeslee B,Greenwalt S A,Butler H J,Hinkley SJ,Zhang L,Rebar E J,Gregory P D,Urnov F D.Precise genome modification in the crop species Zea mays using zinc-finger nucleases.Nature,2009,459:437-411.
[5]Fauser F,Schiml S,Puchta H.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.Plant J,2014,2:348-359.
[6]Li J F,Norville J E,Aach J,McCormack M,Zhang D,Bush J,Church G M,Sheen J.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nat Biotechnol,2013,31:688-691.
[7]Fauser F,Schiml S,Puchta H.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.Plant J,2014,79:348-359.
[8]Feng Z,Zhang B,Ding W,Liu X,Yang DL,Wei P,Cao F,Zhu S,Zhang F,Mao Y,Zhu J K.Efficient genome editing in plants using a CSRIPR/Cas system.Cell Res,2013,23:1229-1232.
[9]Hyun Y B,Kim J,Cho S W,Choi Y,Kim J S,Coupland G.Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted REGN of the CRISPR/Cas9 system to generate heritable null alleles.Planta,2015,241:271-284.
[10]Feng Z,Mao Y,Xu N,Zhang B,Wei P,Yang D L,Wang Z,Zhang Z,Zheng R,Yang L,Zeng L,Liu X,Zhu J K.Multigeneration analysis reveals the inheritance,specificity,and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.Proc Natl Acad Sci USA,2014,111:4632-4637.
[11]Ali Z,Abul-faraj A,Li L,Ghosh N,Piatek M,Mahjoub A,Aouida M,Piatek A,Baltes N J,Voytas D F,Dinesh-Kumar S,Mahfouz M M.Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system.Mol Plant,2015,8:1288-1291.
[12]Gao J,Wang G,Ma S,Xie X,Wu X,Zhang X,Wu Y,Zhao P,Xia Q.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.Plant Mol Biol,2015,87:99-110.
[13]Nekrasov V,Staskawicz B,Weigel D,Jones J D,Kamoun S.Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.Nat Biotechnol,2013,31:691-693.
[14]Shan Q,Wang Y,Li J,Zhang Y,Chen K,Liang Z,Zhang K,Liu J,Xi J J,Qiu J L,Gao C.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol,2013,31:686-688.
[15]Miao J,Guo D S,Zhang J Z,Huang Q P,Qin G J,Zhang X,Wan J M,Gu H Y,Qu L J.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Res,2013,23:1233-1236.
[16]Zhang H,Zhang J,Wei P,Zhang B,Gou F,Feng Z,Mao Y,Yang L,Zhang H,Xu N,Zhu J K.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.Plant Biotechnol J,2014,12:797-807.
[17]Xie K B,Yang Y N.RNA-guided genome editing in plants using a CRISPR-Cas system.Mol Plant,2013,6:1975-1983.
[18]Xu R,Li H,Qin R,Wang L,Li L,Wei P,Yang J.Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.Rice,2014,1:5-7.
[19]Xie K B,Minkenberg B,Yang Y N.Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.Proc Natl Acad Sci USA,2015,112:3570-3575.
[20]Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,Zhao X,Dong Z,Liu Y G.A robust CRISPR/Cas9 system for convenient high-efficiency multiplex genome editing in monocot and dicot plants.Mol Plant,2015,8:1274-1284.
[21]Zhou H,Liu B,Weeks D P,Spalding M H,Yang B.Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice.Nucleic Acids Res,2014,42:10903-10914.
[22]Sun X,Hu Z,Chen R,Jiang Q,Song G,Zhang H,Xi Y.Targeted mutagenesis in soybean using the CRISPR-Cas9 system.Sci Rep,2015,5:10342.
[23]Pan C T,Ye L,Qin L,Liu X,He Y J,Wang J,Chen L F,Lu G.CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations.Sci Rep,2016,6:24765.
[24]Jiang W,Zhou H,Bi H,Fromm M,Yang B,Weeks D P.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice.Nucleic Acids Res,2013,41:e188.
[25]Liang Z,Zhang K,Chen K,Gao C.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.J Genet Genomics,2014,41:63-68.
[26]Li C,Unver T,Zhang B H.A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton(Gossypium hirsutum L.).Sci Rep,2017,7:43902.
[27]Shan Q W,Wang Y P,Li J,Gao C X.Genome editing in rice and wheat using the CRISPR/Cas9 system.Nat Protocol,2014,9:2395-2410.
[28]Li B,Cui G,Shen G,Zhan Z,Huang L,Chen J,Qi X.Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza.Sci Rep,2017,7:43320.
[29]Xu R,Li H,Qin R,Wang L,Li L,Wei P,Yang J.Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.Rice,2014,7:1-5.
[30]Mao Y,Zhang H,Xu N,Zhang B,Gou F,Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants.Mol Plant,2013,6:2008-2011
[31]Xing H L,Dong L,Wang Z P,Zhang H Y,Han C Y,Liu B,Wang X C,Chen Q J.A CRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol,2014,14:327-328.
[32]Li J F,Norville J E,Aach J,McCormack M,Zhang D,Bush J,Church G M,Sheen J.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nat Biotechnol,2013,8:688.
[33]Liu W,Zhu X,Lei M,Xia Q,Botella J R,Zhu J K,Mao Y.A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana.Sci Bull,2015,15:1332-1347.
[34]Wang Y,Cheng X,Shan Q,Zhang Y,Liu J,Gao C,Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol,2014,32:947-951.
[35]Wang F,Wang C,Liu P,Lei C,Hao W,Gao Y,Liu Y G,Zhao K.Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922.PLoSOne,2016,11:e0154027.
[36]Ran F A,Cong L,Yan W X,Scott D A,Gootenberg J S,Kriz A J,Zetsche B,Shalem O,Wu X,Makarova K S,Koonin E V,Sharp P A,Zhang F.In vivo genome editing using Staphylococcus aureus Cas9.Nature,2015,520:186-191.
[37]Kleinstiver B P,Prew M S,Tsai S Q,Topkar V V,Nguyen N T,Zheng Z,Gonzales A P,Li Z,Peterson R T,Yeh J R,Aryee M J,Joung J K.Engineering CRISPR-Cas9 nucleases with altered PAM specificities.Nature,2015,523:481-485.
[38]Hu X X,Wang C,Fu Y P,Liu Q,Jiao X Z,Wang K J.Expanding the range of CRISPR/Cas9 genome editing in rice.Mol Plant,2016,9:943-945.
[39]Hu X X,Meng X B,Liu Q,Li J Y,Wang C J.Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.Plant Biotechnol J,2018,16:292-297.
[40]Hiei Y,Komari T,Kubo T.Transformation of rice mediated by Agrobacterium tumefaciens.Plant Mol Biol,1997,35:205-218.
[41]Wang K,Liu H Y,Du L P,Ye X G.Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.Plant Biotechnol J,2017,15:614-623.