3种方法提取菠萝蜜叶片和种子总RNA的比较
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摘要
为了筛选最适的菠萝蜜总RNA提取方法,采用Trizol提取法、Tris-硼酸提取法和试剂盒提取法提取菠萝蜜叶片和种子总RNA,通过紫外分光光度计检测、琼脂糖凝胶电泳、RT-PCR检测以及Agilent2100检测,对提取结果进行比较分析。结果表明:采用这3种方法均能从菠萝蜜叶片和种子中提取到总RNA,Tris-硼酸提取法较其他2种方法所提取的总RNA产率高,但杂质较多;采用Tris-硼酸提取法能提取完整性较好的总RNA,28S、18S和5S清晰可见,但提取质量较试剂盒提取法差;试剂盒提取法省时省力,有明显的28S和18S条带,A_(260)/A_(280)分布在1.92～1.98,A_(260)/A_(230)分布在2.04～2.09,叶片总RNA产率42.3 ng/μL,种子总RNA产率15.6 ng/μL。RT-PCR检测结果表明在约200 bp处存在清晰整洁的电泳条带,Agilent 2100检测结果显示杂峰极少,28S峰积分面积约是18S峰积分面积的2倍。经综合评定认为,试剂盒提取法是适合菠萝蜜叶片和种子总RNA提取的方法。
In order to screen out the optimal total RNA extracion method from Artocarpus heterophyllus, total RNA was extracted from leaves and seeds in A. heterophyllus by three methods of Trizol extraction, Tris-boric acid extraction and kit extraction. The extraction results were analyzed by UV spectrophotometer, agarose gel electrophoresis, RTPCR and Agilent 2100. The results showed that all of the three methods could extract total RNA from leaves and seeds in A. heterophyllus. Tris-boric acid extraction method had higher total RNA yield than the other two methods, but more impurities. Tris-boric acid extraction method could extract complete total RNA, 28 S, 18 S and 5 S were visible and clear, but quality of extraction was inferior to that of kit extraction method. The kit extraction method was time-saving and labor-saving, extraction had distinct 28 S and 18 S bands, A_(260)/A_(280) was from 1.92 to 1.98, A_(260)/A_(230) was from 2.04 to2.09, total RNA yield in leaves was 42.3 ng/μL, total RNA yield in seeds was 15.6 ng/μL, and there was a clear and tidy electrophoretic band at about 200 bp. The results of Agilent 2100 detection showed very few miscellaneous peak. Integral area of 28 S peak was about 2 times that of 18 S peak. The results of comprehensive evaluation showed that kit extraction method was suitable for total RNA extraction from leaves and seeds in A. heterophyllus.
In order to screen out the optimal total RNA extracion method from Artocarpus heterophyllus, total RNA was extracted from leaves and seeds in A. heterophyllus by three methods of Trizol extraction, Tris-boric acid extraction and kit extraction. The extraction results were analyzed by UV spectrophotometer, agarose gel electrophoresis, RTPCR and Agilent 2100. The results showed that all of the three methods could extract total RNA from leaves and seeds in A. heterophyllus. Tris-boric acid extraction method had higher total RNA yield than the other two methods, but more impurities. Tris-boric acid extraction method could extract complete total RNA, 28 S, 18 S and 5 S were visible and clear, but quality of extraction was inferior to that of kit extraction method. The kit extraction method was time-saving and labor-saving, extraction had distinct 28 S and 18 S bands, A_(260)/A_(280) was from 1.92 to 1.98, A_(260)/A_(230) was from 2.04 to2.09, total RNA yield in leaves was 42.3 ng/μL, total RNA yield in seeds was 15.6 ng/μL, and there was a clear and tidy electrophoretic band at about 200 bp. The results of Agilent 2100 detection showed very few miscellaneous peak. Integral area of 28 S peak was about 2 times that of 18 S peak. The results of comprehensive evaluation showed that kit extraction method was suitable for total RNA extraction from leaves and seeds in A. heterophyllus.
引文
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[25]董璐,贾红梅,刘迪,等.菊花叶片总RNA提取方法的比较研究[J].江苏农业科学,2016,44(3):67-69.
[2]ACEDO A L. Jackfruit biology, production, use, and Philippine research[M]. Arlington:Forestry/FuelwoodResearchand Development, 1992:51.
[3]陈定如.土沉香、木荷、菠萝蜜、构树[J].广东园林,2009,31(3):76-77.
[4]汪永保,余庆,李映志,等.菠萝蜜荧光定量PCR分析中内参基因的筛选[J].热带作物学报,2014,35(7):1374-1381.
[5]董黎梨,汪永保,李映志,等.菠萝蜜果实糖苷酶和多聚半乳糖醛酸酶的活性变化[J].南方农业学报,2013,44(6):924-929.
[6]李映志,董黎梨,汪永保,等.干、湿苞木菠萝果实成熟过程中糖代谢特征[J].果树学报,2014,31(1):85-90.
[7]李莹.菠萝蜜蔗糖合成酶(AhSS2)基因全长序列的克隆及多态性分析[D].湛江:广东海洋大学,2017:51.
[8]李映志,吴成焕,何楚仪,等.用改良CTAB法提取菠萝蜜叶和花序总RNA[J].热带作物学报,2009,30(2):167-169.
[9]段恋.菠萝蜜果锈病发生的生理基础及抗性相关基因分析[D].海口:海南大学,2018:8.
[10]侯哲,陆秀君,张晓林,等.天女木兰不同组织总RNA提取方法的筛选与优化[J].中南林业科技大学学报,2016,36(7):109-115.
[11]荣健,周韬,王平,等.蚬壳花椒种子总RNA提取方法比较与优化[J].中南林业科技大学学报,2017,37(6):60-64.
[12]庞新华,张宇,黄国弟,等.菠萝叶片总RNA提取方法的比较[J].经济林研究,2016,34(1):153-157.
[13]魏琦琦,冯延芝,林青,等.适于转录组测序的枣不同器官总RNA提取方法筛选[J].经济林研究,2015,33(2):63-67.
[14]康亚璇,陈武,庞晓明,等.枣不同组织提取RNA方法的比较及优化[J].分子植物育种,2015,13(12):2871-2882.
[15]牛美云,程庆伟,张月雅,等.檀香心材总RNA提取方法的比较研究[J].热带亚热带植物学报,2017,25(1):20-25.
[16]孙思,陈洁玲,舒灿伟,等.木麻黄小枝总RNA提取方法的比较与优化[J].华中农业大学学报,2014,33(1):51-55.
[17]刘艳娜,李育奇,宋婷婷,等.观赏海棠不同组织中RNA的几种提取方法[J].北京农学院学报,2012,27(4):7-9.
[18]申世刚,高燕霞,王峰山,等.一种梨树组织RNA提取方法[J].河北大学学报(自然科学版),2018,38(4):392-395.
[19]陶倩,范慧艳,张水利,等.南方红豆杉不同组织总RNA提取方法研究[J].中华中医药杂志,2018,33(8):3336-3341.
[20]张力鹏,张银兴,宋文芹,等.红景天属植物叶片RNA高效提取的方法[J].南开大学学报(自然科学版),2017,50(6):48-53.
[21]关玲,赵密珍,王庆莲,等.改良CTAB方法提取果树不同组织的RNA[J].江苏农业科学,2018,46(15):19-22.
[22]罗辉,朱立成.紫藤种子总RNA提取方法研究[J].井冈山大学学报(自然科学版),2017,38(6):35-37.
[23]耿学军,冯紫洲,李晓江,等.沙葱总RNA的提取与质量分析[J].内蒙古民族大学学报(自然科学版),2014,29(6):653-655,719.
[24]徐小勇,许晓玲,张一卉,等.柑橘原生质体总RNA提取方法研究[J].扬州大学学报(农业与生命科学版),2016, 237(2):111-114.
[25]董璐,贾红梅,刘迪,等.菊花叶片总RNA提取方法的比较研究[J].江苏农业科学,2016,44(3):67-69.