CN107058322A - 与杜氏盐藻细胞抗盐能力相关的miRNA - Google Patents
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Abstract
本发明涉及生物技术领域，具体公开了与杜氏盐藻细胞抗盐能力相关的miRNA，其序列选自SEQ ID NO.1～SEQ ID NO.16。可以选取其中的一个或多个进行杜氏盐藻抗盐活动的研究，并且还能够与目前已发现的miRNA一起用于定制芯片，鉴定所述miRNA是否表达，或检测所述miRNA表达量的高低，可用于藻类或植物耐盐抗逆性等研究。
Description
技术领域
本发明属于生物技术领域，具体地说，涉及与杜氏盐藻细胞抗盐能力相关的miRNA。
背景技术
MicroRNA(miRNA)是在真核生物中发现的一类内源性的具有调控功能的非编码RNA，其长度约为21-25个核苷酸。成熟miRNA的产生是由较长的初级转录本(pri-miRNA)经过一系列核酸酶的剪切加工而成的，随后组装进RNA诱导的沉默复合体，通过碱基互补配对的方式识别靶mRNA，并根据互补程度的不同指导沉默复合体降解靶mRNA或者阻遏靶mRNA的翻译。研究表明miRNA参与了生命过程中多种重要进程，包括发育、细胞增殖和凋亡、细胞分化、脂肪代谢等等。目前在miRBase数据库中收录有人(Homo sapiens)的pre-miRNA(miRNA前体)1881条，小鼠(Mus musculus)有1193条，大鼠(Rattus norvegicus)有495条，莱茵衣藻(Chlamydomonas reinhardtii)有50条，而在杜氏盐藻(Dunaliella salina，D.salina)中发现的miRNA少之又少。杜氏盐藻是一种比较独特的高度耐盐单细胞真核生物，属绿藻门绿藻纲团藻目多睫毛科。杜氏盐藻细胞没有细胞壁，具双鞭毛而能游动，含有一个较大的杯状叶绿体能进行光合作用；可在含有0.05M-5M NaCl的盐水中生长，抗逆性极佳，是一种较为理想的抗逆境生物。杜氏盐藻细胞因含有丰富的β-胡萝卜素而具有独特经济价值。极其耐盐的能力、简单的细胞结构、便利的培养条件及丰富的营养价值使杜氏盐藻成为进行生物能源、耐盐抗逆性等研究的重要模式生物和遗传学研究的良好材料。miRNA具有物种和组织特异性，最近几年关于植物miRNA的各类研究越来越多，例如抗旱、抗盐等抗逆性研究。
发明内容
本发明以杜氏盐藻为研究对象，发现了一些在miRBase数据库中未被收录的预测miRNA，其核苷酸序列如SEQ ID NO.1-16所示。经试验证实，其与杜氏盐藻细胞抗盐能力相关。
因此，本申请提供了新的与抗盐胁迫相关的miRNA序列。
在此基础上，所述miRNA的前体序列及其前体序列的编码序列也属于本发明的保护范围。
经试验研究发现，前述miRNA的表达量受到盐胁迫的调节，且调节效果明显，说明其在杜氏盐藻适应高盐胁迫机制中发挥重要作用。
所述试验研究具体为，通过对高盐(含3.0M NaCl)和低盐(含0.75M NaCl)培养条件下测序获得的杜氏盐藻样品小RNA序列进行数据分析并采用Fisher精确检验分析发现，上述miRNA与杜氏盐藻细胞的抗盐能力显著相关。具体表现为：相对在低盐环境中的表达量，上述其中一条miRNA(SEQ ID NO.1)在高盐环境中的表达量显著降低(P<0.05)，其余15条miRNA(SEQ ID NO.2-16)在高盐环境中的表达量显著升高(P<0.05)。
因此，本发明进一步提供了所述miRNA在杜氏盐藻抗盐胁迫中的应用，以及其在培育耐盐转基因植物中的应用，具体为在所述转基因植物中过表达所述miRNA或抑制所述miRNA的表达。
更进一步地，基于前述研究及发现，检测本发明所述miRNA的试剂也应当属于本发明的保护范围，所述试剂可用于判断/检测杜氏盐藻细胞的抗盐能力。
不仅如此，基于前述研究及发现，可从本发明提供的miRNA(SEQ ID NO.1-16)中单独取其中一个/多个进行抗盐胁迫研究，并且还能够联合目前已发现的miRNA一起用于定制芯片，用于藻类或植物耐盐抗逆性等研究。
因此，本发明还提供了一种miRNA芯片，其可鉴定本发明所述的miRNA是否表达，或检测本发明所述miRNA表达量的高低，但并不限于仅检测本发明所提供的miRNA。应当理解的是，只要所述miRNA芯片可用于检测本发明所述的miRNA，该miRNA芯片均属于本发明的保护范围。
本发明的有益效果在于：
本发明发现并提供了新的与杜氏盐藻细胞抗盐能力相关的miRNA，可以选取其中一个或多个进行杜氏盐藻抗盐活动的研究，并且还能够与目前已发现的miRNA一起用于定制芯片，用于藻类或植物耐盐抗逆性等研究。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、杜氏盐藻细胞总RNA的提取鉴定
确定杜氏盐藻细胞生长状态良好，收集培养至对数生长期的杜氏盐藻细胞。将杜氏盐藻细胞培养液移入离心管中，离心沉淀细胞，弃去上清液。按照RNAiso Plus[宝生物工程(大连)有限公司，中国]说明提取杜氏盐藻细胞总RNA。将总RNA进行液相质谱(LC)检测并根据质量评价标准判断提取的总RNA质量是否满足后续实验要求。评价结果表明，其OD260/280比值≥1.8，RIN值≥7且rRNA 28S/18S比值≥0.7，以上结果说明提取的总RNA质量较好，满足实验要求。
实施例2、小RNA测序文库的构建和测序实验
实验流程按照Illumina公司提供的标准步骤执行，包括制备文库和测序实验。小RNA测序文库制备采用TruSeq Small RNA Sample Prep Kits(Illumina，San Diego，USA)试剂盒。杜氏盐藻细胞样本提取总RNA后，利用miRNA的属性，即5'端为磷酸基团，3'端为羟基基团。首先使用T4RNA连接酶2将一个腺苷化单链DNA 3'接头和5'接头相继连接到smallRNA上，其中3'端接头的5端为rAPP，3端有氨基保护，所以能够减少miRNA的自连，T4RNA连接酶2(Truncated)在连接反应时不需要ATP，只需要预腺苷化的接头，所以能够减少miRNA以及接头序列的自连。5'端接头被设计成可以捕获带有5'磷酸基团的small RNA。带有5'与3'连接接头的small RNA序列，通过与3'端互补的RT引物进行反转录反应，最后对反转录产生的cDNA序列进行PCR扩增。最后，对140-160bp长度范围的PCR产物进行6％polyacrylamideTris-borate-EDTA 胶回收，从而完成整个文库的制备工作。对构建好的文库使用Illumina Hiseq 2500进行测序，测序读长为单端1X50bp。
实施例3、数据生物信息学分析发现新的miRNA
应用联川生物公司所提供miRNA数据分析软件ACGT101-miR(LC Sciences，Houston，Texas，USA)实施例2获得的测序数据进行分析处理。其总体分析流程简言之，即原始序列通过IlluminaFastQC进行数据质量评估，获取Q30数据后，通过一系列数据处理，将由于样本制备、测序接头、非典型miRNA特征序列(在此我们称为垃圾序列)以及测序仪器光学数码处理而产生的非纯序列(N特征序列)进行清理；随后，进行长度筛选，保留碱基长度在18-25nt(植物)，再将剩余序列比对各种RNA数据库序列(不包含miRNA)，如mRNA、RFam(包含rRNA，tRNA，snRNA，snoRNA等)和重复序列数据库(Repbase)，并进行过滤。将上述清理、去除过后所剩余的测序序列，通过Bowtie比对到miRBase 21.0中特定物种的前体上，鉴定该物种已知的miRNA，同时发现新的5p或者3p miRNA序列。在比对分析中，5'和3'末端长度变化以及序列内部有一个错配的情况，在比对中被允许。其中，比对到该特定物种的成熟体序列部分miRNA即为已知报道的miRNA，而如果检测到的序列能够比对到已知miRNA的对应臂部位则可以被确定为新的5p或者3p miRNA候选序列。未比对上的序列，再一次通过Bowtie比对到miRBase 21.0中其它选定物种的前体上，并且比对上的miRNA前体序列通过进一步比对上测序物种的基因组序列从而鉴定出该类已报道miRNA。由于以上两类鉴定出的miRNA都来自miRBase数据库已报道的序列，因而我们定义为已知的miRNA。其它未比对到所选物种的前体的序列，使其进一步与基因组比对，对于比对上的序列，则在基因组上相应比对位点，通过碱基数目延伸(植物120nt)，并采用RNAfold软件(http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi)进行RNA二级结构预测。最终得到16个尚未收录于miRBase数据库的预测的miRNA，命名为SEQ ID NO.1～SEQ ID NO.16。
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<110> 新乡医学院
<120> 与杜氏盐藻细胞抗盐能力相关的miRNA
<130> KHP171110216.0TQ
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> miRNA
<400> 1
ttgcagacaa tgtcaagggc c 21
<210> 2
<211> 21
<212> DNA
<213> miRNA
<400> 2
taggatcagc agcaacgcat g 21
<210> 3
<211> 21
<212> DNA
<213> miRNA
<400> 3
tgggtgttgg tagatgcagt t 21
<210> 4
<211> 21
<212> DNA
<213> miRNA
<400> 4
tgtttgctgg aagacgtgtg t 21
<210> 5
<211> 21
<212> DNA
<213> miRNA
<400> 5
tgcttgctat tctctgagaa t 21
<210> 6
<211> 21
<212> DNA
<213> miRNA
<400> 6
tcatgtacac atgcctggaa t 21
<210> 7
<211> 21
<212> DNA
<213> miRNA
<400> 7
ttcagcagat ggtcaaggac t 21
<210> 8
<211> 20
<212> DNA
<213> miRNA
<400> 8
ttctgtaact gtgcatgtta 20
<210> 9
<211> 21
<212> DNA
<213> miRNA
<400> 9
tcatgttcac atgcctggat t 21
<210> 10
<211> 21
<212> DNA
<213> miRNA
<400> 10
tgaacttcat cttgatcctc a 21
<210> 11
<211> 21
<212> DNA
<213> miRNA
<400> 11
tcagtttctc atgacaagaa t 21
<210> 12
<211> 21
<212> DNA
<213> miRNA
<400> 12
tcgttaatat tgttaaagat g 21
<210> 13
<211> 22
<212> DNA
<213> miRNA
<400> 13
tgtgctccac atactgccag ag 22
<210> 14
<211> 21
<212> DNA
<213> miRNA
<400> 14
tctcgtctgg gtattgtgag t 21
<210> 15
<211> 21
<212> DNA
<213> miRNA
<400> 15
ttctgaaaag tcaagggctc t 21
<210> 16
<211> 20
<212> DNA
<213> miRNA
<400> 16
tggacatgat ccgcgcgtgg 20
Claims (9)
1.与杜氏盐藻细胞抗盐能力相关的miRNA，其特征在于，其序列选自SEQ ID NO.1～SEQID NO.16。
2.权利要求1所述miRNA的前体序列。
3.编码权利要求2所述前体序列的DNA序列。
4.权利要求1所述的miRNA在杜氏盐藻抗盐胁迫中的应用。
5.权利要求1所述的miRNA在培育耐盐转基因植物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述转基因植物中过表达所述miRNA或抑制所述miRNA的表达。
7.检测权利要求1所述的miRNA的试剂。
8.权利要求7所述的试剂在判断/检测杜氏盐藻细胞抗盐能力方面的应用。
9.一种miRNA芯片，其特征在于，鉴定权利要求1所述的miRNA是否表达，或检测权利要求1所述miRNA表达量的高低。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880745A (zh) * | 2019-03-15 | 2019-06-14 | 江苏大学 | 一种利用腌制废水、晒盐卤水分段培养盐藻的方法 |
| CN113549620A (zh) * | 2021-07-13 | 2021-10-26 | 山西大学 | 多型杜氏藻盐胁迫响应miRNAs及其应用 |
-
2017
- 2017-04-26 CN CN201710284159.4A patent/CN107058322A/zh active Pending
Non-Patent Citations (2)
| Title |
|---|
| LI JINGRUI 等: "microRNAs in a multicellular green alga Volvox carteri", 《SCIENCE CHINA LIFE SCIENCES》 * |
| 娄素琳 等: "盐藻 microRNAs 高通量测序和生物信息学分析", 《深圳大学学报理工版》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880745A (zh) * | 2019-03-15 | 2019-06-14 | 江苏大学 | 一种利用腌制废水、晒盐卤水分段培养盐藻的方法 |
| CN113549620A (zh) * | 2021-07-13 | 2021-10-26 | 山西大学 | 多型杜氏藻盐胁迫响应miRNAs及其应用 |
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Application publication date: 20170818 |