CRISPR/Cas9介导的番茄SlMAPK6突变对植株形态的影响
发布时间：2021-09-23 04:13
促分裂原活化蛋白激酶（MAPK）级联信号参与调控植物的多种逆境、生长发育以及花青苷的合成通路。为探究SlMAPK6在番茄中的功能,本研究利用CRISPR/Cas9技术设计3个靶标位点定点编辑SlMAPK6,并获得突变体CRISPR-3和CRISPR-7。PCR和测序结果发现SlMAPK6敲除载体构建成功。通过对T1植株进行卡那霉素基因筛选、靶位点扩增测序及行脱靶效应分析,结果发现矮番茄植株成功突变且未脱靶,与野生型对照表型相比,所有突变植株表现出根系更发达、侧枝增多的表型,表明SlMAPK6在番茄植株形态建成的过程中发挥了重要作用,为进一步探究SlMAPK6在番茄生长发育中的作用奠定了基础。
【文章来源】：核农学报. 2020,34(09)北大核心CSCD
【文章页数】：8 页
【部分图文】：
SlMAPK6基因图示与靶点位置和序列
表 1 试验中所用的引物Table 1 The primers used in this study 目的Aim 引物名称Primer name 引物序列(5′-3′)Primer sequence(5′-3′) 产物大小Product size/bp 检测靶标位点突变Detection of target site mutation Target1-F CAAGATTCTTCAACCCCTTTTCT 448 Target1-R CACGACCAATAGGTCTGATAGGA Target2-F GAAGATTGGGAATGCATTTGATA 390 Target2-R AACAAGGCAAAGACATTTGAAGA Target3-F ATGTTATACGGCCTCCTCAAAAG 386 Target3-R CCTGAAAAGTGTTTACCGCTATG 卡那霉素抗性基因检测Kan resistance gene test Kan-F GGCGATACCGTAAAGCACGA 728 Kan-R ATGGATTGCACGCAGGTTCT 潜在脱靶位点验证Verification of potential off-target sites Off-target1-F CCATGATGTGTGGGATCTTCT 238 Off-target1-R CAGGACAGTGACCATCCCTAA Off-target2-F GCTCGAGTTTGTTCCGAATC 222 Off-target2-R CACTTCCTCCTGAACCCTGA Off-target3-F TTTTTCTTCAGTGCCGCTTT 196 Off-target3-R TGCACAAGGTACAAGGCAGT Off-target4-F TGCTTTCACATTGCCAGGTA 241 Off-target4-R CAGGCACTGCATTACCATCA表2 潜在脱靶序列的突变Table 2 Potential off-target sites 潜在脱靶位点Potential off-target sites 潜在脱靶位点序列Potential off-target sequence 错配的碱基数No. of mismatch bases 检测的植株数No. of plants tested 突变体数No. of mutations T1 GGTGAAAACAGTAACATAAAAGG 4 30 0 T2 CAAATAGCTTATCAGTCACAGGG 4 30 0 T3 GATAAAGCTTCTTCGTCACATGG 2 30 0 T4 AGGTCTCATAAGTCAGCTGTTGG 4 30 0 注:划线标记为PAM序列(NGG),错配的碱基用斜体表示。Note: PAM sequence (NGG) was underlined, mismatch bases were indicated in italic.
表3 T1野生型(WT)及突变体(CRISPR-3和CRISPR-7)表型数据对比Table 3 Comparison of phenotypic data of wild-type and mutant (CRISPR-3 and CRISPR-7) in T1 genegration 时期Stage 植株类型Plant stye 侧枝数Number branch 不定根数Number of adventitious roots 根长Root length/cm 茎径Stem diameter/cm 幼苗期Seedling stage WT 6.33±0.58c 0b 5.78±0.35b 0.13±0.04b CRISPR-3 10.67±1.15b 21.00±11.36a 12.31±2.54a 0.24±0.02a CRISPR-7 12.67±0.58a 14.67±9.07b 10.51±2.27a 0.24±0.03a 成熟期Maturation stage WT 12.67±0.58c 0c 26.82±1.65b 0.33±0.01b CRISPR-3 21.67±0.58a 64.67±7.51a 46.01±1.59a 0.40±0.01a CRISPR-7 19.67±1.53b 46.00±7.81b 43.30±0.87a 0.40±0.02a 注:同列不同小写字母表示同一时期不同植株类型之间差异显著(P<0.05)。Note: Different lowercase letters in the same column indicate significant differeces at 0.05 level among different plant stye in same stage.3 讨论
【参考文献】：
期刊论文
[1]利用CRISPR/Cas9基因编辑技术敲除水稻NRR基因促进根系生长的研究[J]. 王海明,张立强,李娜,刘建丰,马崇烈. 杂交水稻. 2019(05)
[2]利用CRISPR/Ca9技术靶向编辑芥蓝BoaZDS[J]. 郑爱红,张芬,江敏,袁巧,江雷雨,陈清,汤浩茹,孙勃. 园艺学报. 2019(01)
[3]利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究[J]. 郭晔,万东艳,柴壮壮,王跃进,文颖强. 园艺学报. 2019(04)
[4]CRISPR/Cas9定点编辑水稻LOCOs05g31750基因位点及靶修饰位点遗传稳定性研究[J]. 沈春修,却志群,刘莹,廖锦风. 核农学报. 2018(06)
[5]不同根型水稻的根系可塑性比较研究[J]. 楼珏,杨文清,杨玲,李铁梅,卢华金,楼巧君. 核农学报. 2018(06)
[6]Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton[J]. Yanling Wang,Zhigang Meng,Chengzhen Liang,Zhaohong Meng,Yuan Wang,Guoqing Sun,Tao Zhu,Yongping Cai,Sandui Guo,Rui Zhang,Yi Lin. Science China（Life Sciences）. 2017(05)
[7]番茄SlMAPK9-2基因分离及表达分析[J]. 王洁,王燕,潘长田,何艳军,刘雪,卢钢. 核农学报. 2016(08)
[8]Genome engineering using the CRISPR/Cas system[J]. Takuro Horii,Izuho Hatada. World Journal of Medical Genetics. 2014(03)
博士论文
[1]生长素及独脚金内酯介导H2O2调控番茄侧枝生长发育的机制研究[D]. 陈小娟.浙江大学 2015
[2]番茄SpMPKs基因响应非生物胁迫的功能分析[D]. 李翠.西北农林科技大学 2014
本文编号：3405015
【文章来源】：核农学报. 2020,34(09)北大核心CSCD
【文章页数】：8 页
【部分图文】：
SlMAPK6基因图示与靶点位置和序列
表 1 试验中所用的引物Table 1 The primers used in this study 目的Aim 引物名称Primer name 引物序列(5′-3′)Primer sequence(5′-3′) 产物大小Product size/bp 检测靶标位点突变Detection of target site mutation Target1-F CAAGATTCTTCAACCCCTTTTCT 448 Target1-R CACGACCAATAGGTCTGATAGGA Target2-F GAAGATTGGGAATGCATTTGATA 390 Target2-R AACAAGGCAAAGACATTTGAAGA Target3-F ATGTTATACGGCCTCCTCAAAAG 386 Target3-R CCTGAAAAGTGTTTACCGCTATG 卡那霉素抗性基因检测Kan resistance gene test Kan-F GGCGATACCGTAAAGCACGA 728 Kan-R ATGGATTGCACGCAGGTTCT 潜在脱靶位点验证Verification of potential off-target sites Off-target1-F CCATGATGTGTGGGATCTTCT 238 Off-target1-R CAGGACAGTGACCATCCCTAA Off-target2-F GCTCGAGTTTGTTCCGAATC 222 Off-target2-R CACTTCCTCCTGAACCCTGA Off-target3-F TTTTTCTTCAGTGCCGCTTT 196 Off-target3-R TGCACAAGGTACAAGGCAGT Off-target4-F TGCTTTCACATTGCCAGGTA 241 Off-target4-R CAGGCACTGCATTACCATCA表2 潜在脱靶序列的突变Table 2 Potential off-target sites 潜在脱靶位点Potential off-target sites 潜在脱靶位点序列Potential off-target sequence 错配的碱基数No. of mismatch bases 检测的植株数No. of plants tested 突变体数No. of mutations T1 GGTGAAAACAGTAACATAAAAGG 4 30 0 T2 CAAATAGCTTATCAGTCACAGGG 4 30 0 T3 GATAAAGCTTCTTCGTCACATGG 2 30 0 T4 AGGTCTCATAAGTCAGCTGTTGG 4 30 0 注:划线标记为PAM序列(NGG),错配的碱基用斜体表示。Note: PAM sequence (NGG) was underlined, mismatch bases were indicated in italic.
表3 T1野生型(WT)及突变体(CRISPR-3和CRISPR-7)表型数据对比Table 3 Comparison of phenotypic data of wild-type and mutant (CRISPR-3 and CRISPR-7) in T1 genegration 时期Stage 植株类型Plant stye 侧枝数Number branch 不定根数Number of adventitious roots 根长Root length/cm 茎径Stem diameter/cm 幼苗期Seedling stage WT 6.33±0.58c 0b 5.78±0.35b 0.13±0.04b CRISPR-3 10.67±1.15b 21.00±11.36a 12.31±2.54a 0.24±0.02a CRISPR-7 12.67±0.58a 14.67±9.07b 10.51±2.27a 0.24±0.03a 成熟期Maturation stage WT 12.67±0.58c 0c 26.82±1.65b 0.33±0.01b CRISPR-3 21.67±0.58a 64.67±7.51a 46.01±1.59a 0.40±0.01a CRISPR-7 19.67±1.53b 46.00±7.81b 43.30±0.87a 0.40±0.02a 注:同列不同小写字母表示同一时期不同植株类型之间差异显著(P<0.05)。Note: Different lowercase letters in the same column indicate significant differeces at 0.05 level among different plant stye in same stage.3 讨论
【参考文献】：
期刊论文
[1]利用CRISPR/Cas9基因编辑技术敲除水稻NRR基因促进根系生长的研究[J]. 王海明,张立强,李娜,刘建丰,马崇烈. 杂交水稻. 2019(05)
[2]利用CRISPR/Ca9技术靶向编辑芥蓝BoaZDS[J]. 郑爱红,张芬,江敏,袁巧,江雷雨,陈清,汤浩茹,孙勃. 园艺学报. 2019(01)
[3]利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究[J]. 郭晔,万东艳,柴壮壮,王跃进,文颖强. 园艺学报. 2019(04)
[4]CRISPR/Cas9定点编辑水稻LOCOs05g31750基因位点及靶修饰位点遗传稳定性研究[J]. 沈春修,却志群,刘莹,廖锦风. 核农学报. 2018(06)
[5]不同根型水稻的根系可塑性比较研究[J]. 楼珏,杨文清,杨玲,李铁梅,卢华金,楼巧君. 核农学报. 2018(06)
[6]Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton[J]. Yanling Wang,Zhigang Meng,Chengzhen Liang,Zhaohong Meng,Yuan Wang,Guoqing Sun,Tao Zhu,Yongping Cai,Sandui Guo,Rui Zhang,Yi Lin. Science China（Life Sciences）. 2017(05)
[7]番茄SlMAPK9-2基因分离及表达分析[J]. 王洁,王燕,潘长田,何艳军,刘雪,卢钢. 核农学报. 2016(08)
[8]Genome engineering using the CRISPR/Cas system[J]. Takuro Horii,Izuho Hatada. World Journal of Medical Genetics. 2014(03)
博士论文
[1]生长素及独脚金内酯介导H2O2调控番茄侧枝生长发育的机制研究[D]. 陈小娟.浙江大学 2015
[2]番茄SpMPKs基因响应非生物胁迫的功能分析[D]. 李翠.西北农林科技大学 2014
本文编号：3405015
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