为鉴定醛脱羰基酶(ECERIFERUM1,CER1)基因在旱生植物表皮蜡质烷烃合成过程中的功能,培育适宜干制加工品种,选定扁果枸杞(Lycium barbarum ssp. Bianguo)LbCER1基因1 469～1 840 bp区段为最佳RNA干扰(RNA interference,RNAi)靶序列,克隆该区段并将其插入pKANNIBAL载体, 构建具有发卡状反向重复序列的RNAi中间载体pKANNIBAL-LbCER1(+)-PDK intron-LbCER1(-),并将发卡状反向重复序列转入RNAi表达载体pART27,再用冻融法将pART27-LbCER1(+)-PDK intron-LbCER1(-)载体转化根癌农杆菌(Agrobacterium tumefaciens) GV3101,并用限制性内切酶双酶切法和PCR法检测转化的农杆菌菌株。结果表明,RNAi的中间载体和表达载体均结构正确,并且RNAi表达载体pART27-LbCER1(+)-PDK intron-LbCER1(-)成功转化根癌农杆菌GV3101,说明该构建方法正确,将为进一步筛选LbCER1- RNAi植株,获得适宜干制加工的宁夏枸杞新品种奠定基础。
Aldehyde decarbonylase is a class of enzymes participate in plant alkane biosynthesis. In order to breed new varieties for fruits drying process, ECERIFERUM1 (CER1), an aldehyde decarbonylase coding gene was subjected into an RNA interference (RNAi) assay in xerophytic plant Lycium barbarum ssp. Bianguo. The segment between the 1,469 bp to 1,840 bp of LbCER1 coding aldehyde decarbonylase region in Lycium barbarum ssp. Bianguo was chosen as the optimal target of RNAi. An intermediate vector, pKANNIBAL-LbCER1(+)-PDK intron-LbCER1(-), was used to achieve the proper hairpin structure required for RNAi assay. The final hairpin sequence containing reverse complementary fragments of the chosen LbCER1 sequence flanking the PDK intron was inserted into the vector pART27. In addition, the final vector, pART27-LbCER1(+)-PDK intron-LbCER1(-), was chemically transformed into Agrobacterium tumefaciens GV3101. Restriction enzyme digestion and PCR methods were employed to screen positive intermediate and final vectors. The result showed that A. tumefaciens containing pART27-LbCER1(+)-PDK intron-LbCER1(-) was achieved. Hence, using this method to generate plasmids was correct, which laid the foundation for obtaining new varieties for dry processing through bioengineering of LbCER1- RNAi plants.
[1] 肖佳,高昊,周正群,等.枸杞属中枸杞红素类成分研究进展[J].科学通报, 2017,62(16):1 691-1 698.
[2] 章中.枸杞保藏加工技术现状[J].中国食物与营养,2008(5):31-33.
[3] 杨爱梅,吴古飞,杜静,等.枸杞表皮蜡质层成分及显微结构研究[J].食品工业科技,2011,32(12):112-114.
[4] BOURDENX B,BEMARD A,DOMERGUE F,et al.Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses[J].Plant Physiology,2011,156(1):29-45.
[5] 袁惠君,马倩国,高泽,等.扁果枸杞角质层蜡质合成相关基因LbCER1的克隆及其表达特征分析[J].华北农学报,2019,34 (5):15-22.
[6] FIRE A,XU S,MONTGOMERY M,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6 669):806-811.
[7] 马建,刘艺苓,王丕武.植物RNA干扰的研究进展[J].中国油料作物学报,2008,30(2):252-259.
[8] ELBASHIR S M,LENDECKEL W T,TUSCHL T.RNA interference is mediated by 21- and 22- nueleotide RNAs[J].Genes & Development,2001,15:188-200.
[9] KERSCHEN A,NAPOLI C A, RICHAD A,et al.Effectiveness of RNA interference in transgenic plants[J].FEBS Letters,2004,566(1-3): 223-228.
[10] WESLEY S V,HELLIWELL C A,SMITH N A,et al.Construct design for efficient effective and high-throughput gene silencing in plants[J].The Plant Journal,2001,27(6):581-590.
[11] BRUMMELKAMP T R,BERNARDS R,AGAMI R.A system for stable express ion of short interfering RNAs in marnrnalian[J].Science.2002,296(5 567):550-553.
[12] DALMAY T,HAMILTON A J,RUDD S.An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus[J].Cell,2000,101(5):543-553.
[13] 陈悦,陈宁美,松布尔巴图,等.盐芥WBC11克隆与RNAi载体的构建[J].中央民族大学学报(自然科学版),2018,27 (1):89-94.
[14] 刘颖,刘国选,黄川腾,等.麻疯树JcFATA基因干涉表达载体构建和功能初步分析[J].农业生物技术学报,2018,26(5):175-186.
[15] 尹国,路正营,李世云,等.利用RNAi研究棉花SSH文库上调表达EST的功能[J].华北农学报,2018,33(1):71-79.
[16] 王益民,张珂,许飞华,等.不同品种枸杞子营养成分分析及评价[J].食品科学,2014,35(1):34-38.
[17] 袁惠君,刘轲,王春梅,等.两个宁夏枸杞品种的耐渗透胁迫和耐盐特征比较[J].草业科学,2016,33(4):681-690.
[18] LIN J H,LEVIN H L.Reverse transcription of a self-primed retro-transposon requires an RNA structure similar to the U5-IR stem-loop of retroviruses[J].Molecular and Cellular Biology,1998,18(11):6 859-6 869.
[19] HAMILTON A J,BAULCOMBE D C.A species of small antisense RNA in posttranscriptional gene silencing in plants[J].Science,1999,286(5 441):950-952.
[20] SMITH N A,SINGH S P,WANG M B.Gene expression: Total silencing by intron-spliced hairpin RNAs[J].Nature,2000,407(6 802):319-320.
[21] MITSUHARA I,SHIRASAWA-SEO N,IWAI T,et al.Release from post-transcriptional gene silencing by cell proliferation in transgenic tobacco plants: Possible mechanism for noninheritance of the silencing[J].Genetics,2002,160(1):343-352.
[22] KLAHRE U,CRETE P, LEUENBERGER S A,et al.High molecular weight RNAs and small interfering RNAs induces systemic posttranscriptional gene sileneing in plants[J].Proceedings of the National Academy of Sciences of the United States of America,2002, 99(18):11 981-11 986.
[23] HAMMOND S M,BERSTEIN E,BEACH D,et al.An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells[J].Nature,2000,404:293-296.
[24] BERNSTEIN E,CAUDY A,HAMMOND S M,et al.Role for a bidentate ribonucleasein the initiation step of RNA interference[J].Nature.2001,409(6 818):363-366.
