摘要
日本落叶松(Larix leptolepis)是我国北方重要的针叶速生用材造林树种，已经在选择育种和杂交育种等方面取得重要进展，然而仍不能满足发展林木良种、提高林木产量、增强落叶松生产力的重大需求。究其原因，除背景复杂、生长周期长等特有问题外，我国针叶树基础理论与分子遗传研究基础薄弱也是导致落叶松改良进程缓慢的原因之一。新型的microRNA(miRNA)在生物生长发育过程中发挥关键调控作用，是农艺性状关键调控因子，正作为手段运用于品种遗传改良研究。本研究以2年生日本落叶松实生苗根、茎、叶总RNA等量混合样品构建small RNA(sRNA)文库，利用Solexa测序平台，结合生物信息学方法筛选调控落叶松生长发育过程的miRNAs；通过psRNATarget软件在线预测miRNAs目标基因；采用qRT-PCR技术研究种子萌发不同时期的种胚(0d、1d、5d、9d、28d)、植株生长不同时期的针叶及茎(90d、1.5a、5a、10a、25a和50a)中miRNA的差异表达及5′RACE技术验证3个目标基因的切割位点。研究结果如下：
(1)利用Illumina GAIIx平台进行sRNA库Solexa测序并筛选出候选miRNAs。获得27,042,027条原始序列，经去噪、除杂，获得长度范围在16nt～26nt的待分析序列7,466,370条，测序数据已提交至NCBI-Gene Expression Omnibus (GEO)数据库，登录号为GSE34805。21nt长度的序列占sRNA的48.39%，表明落叶松sRNA以21nt长度序列为主要类型；将待分析序列与miRBase (17.0)及本室落叶松转录组数据进行BLAST序列比对，获得308个候选miRNAs。Solexa技术成功获得大量针叶树候选miRNAs序列。
(2)利用BLAST比对及二级结构预测鉴定出85个家族的174个miRNAs。77个miRNAs(61.04%为21nt；66.23%以U为5′端首位碱基)属于48个保守家族，占所鉴定的miRNA基因家族总数的56.47%；97个miRNAs属于37个新发现的基因家族(74.23%为21nt；48.45%以U为5′端首位碱基)。48个保守miRNAs基因中有20个是陆地植物共同保守的(miR156、miR159、miR160、miR162、miR164、miR166～miR169、miR171、miR172、miR390、miR393～miR399和miR408)，12个miRNAs仅在针叶树种中特异表达(miR946、miR947、miR950、miR951、miR1311～miR1314、miR1316、miR3697、miR3701和miR3707)。利用qRT-PCR方法对34个保守的和6个新发现的miRNAs进行了表达验证。研究发现和鉴定的miRNA基因，拓宽了我们对针叶树miRNA的认识并极大地丰富了其信息资源。
(3)利用生物信息学手段预测、分析出236个miRNA目标基因。psRNATarget软件预测目标基因及GO蛋白功能注释结果表明，日本落叶松85个miRNA基因家族的236个目标基因主要参与转录调控、生长发育、蛋白转运、胁迫响应、激素信号传导及电子传递等生物过程。
(4)29个miRNAs在种子萌发前期总体呈下降趋势、8个miRNAs在植株生长过程中随植株年龄增加呈规律性表达变化模式。运用qRT-PCR方法分析了miR156等29个miRNAs在日本落叶松种子萌发过程5个关键时期(0d、1d、5d、9d和28d)和植株生长6个时期(90d、1.5a、5a、10a、25a和50a)的表达变化模式。结果显示，miRNAs的表达水平在成熟种子(0d)中最高，吸胀(1d)和萌动(5d)阶段迅速下降，44.83%(13/29)的miRNAs总体呈逐渐降低趋势；种子萌发各个时期，miR894的相对表达量最高，miR408最低。8个miRNAs (miR156、miR172、miR395、miR396、miR397、miR398、miR408和miR947)随年龄增长呈现出规律性上升或下降趋势。5a和25a是许多miRNAs表达变化的转折点，可能是miRNA调控落叶松生长发育的关键时期。研究揭示了在种子萌发和植株生长过程中，miRNAs的差异表达特性可能对落叶松生长发育起着重要调控作用。
(5) APm、EF1和eIF三个适合目标基因qRT-PCR定量研究的内参基因的筛选。利用geNorm和NormFinder两种分析软件对12个候选内参基因的稳定性进行评价，并从中筛选出在落叶松不同器官、不同生长发育时期（体细胞胚胎原胚团时期到胚胎成熟时期、种子萌发过程、植株幼年生长阶段到成年生长阶段）表达稳定的APm、EF1和eIF三个内参基因。为落叶松miRNA目标基因的qRT-PCR表达分析提供最适内参。
(6)落叶松针叶中miR172与其目标基因AP2L1的表达变化在植株生长过程中呈显著负调控关系(r＝–0.960，P=0.002)。miR172、miR397和miR398分别切割其目标基因AP2L1、JR147962(Laccase)和JR143920(Phytocyanin)的mRNA，切割位点位于miRNA与其目标基因互补序列第10和11位碱基之间。miR397、miR398与其目标基因JR147962(Laccase)、JR143920(Phytocyanin)之间的表达变化未发现明显相关性。
上述研究表明，来自85个家族的174个miRNAs中，有8个miRNAs在落叶松的生长发育过程中可能具有重要调控作用。miR172在植株生长过程中与其目标基因AP2L1呈现显著的负相关性。AP2L1作为一种重要的调控日本落叶松植株个体发育过程的功能分子展开研究极具价值。为进一步研究针叶树miRNAs在生长发育过程中的作用及人工定向遗传改良的分子调控机制提供依据。
Japanese larch (Larix leptolepis) is one of the most important coniferous species withfast-growing speed. It is widely used for forestation and production of timber in north China.Some major progress has been achieved upon selective breeding and cross breeding in larch.However, the progress can not meet the significant needs of fine tree varieties development,wood yield improvement and productivity enhancement. Apart from the complicated geneticbackground and long growth cycle, the lack of important breakthroughs in basic theory andmolecular genetic research for coniferous trees has hampered the development of geneticimprovement progress. MicroRNA (miRNA), generally21–24nt in length, spatiotemporallyregulates gene expression at transcriptional and/or post-transcriptional level. MiRNAs andtheir target genes involve in morphogenesis of organs, signal transductions and biotic/abioticstress responses, playing an important role in plant growth and development, nutrienthomeostasis. It is that the central role that miRNA networks play in the control of keyargonomic traits makes them appealing biotechnological targets for the production of varietieswith improved performance. The study on the miRNA identification and function in larch isvery important, not only enriching the information resource of miRNA, but also providing anew idea for the genetic improvement of conifers.
A pooled library was constructed with equal amounts total RNA (10μg) of needle, stem,and root from a two-year-old larch seedling in the present study. Solexa sequencing combiningwith bioinformatics methods were applied for screening and processing the miRNAs whichcould regulate the growth and developement of Japanese larch. The psRNATarget onlinesoftware was used to predict and analyze miRNAs target genes. Quantitative real-time PCR(qRT-PCR) technology was emploied to analyze the differential expression of miRNA duringthe different periods of seed germination (0d、1d、5d、9d、28d) and different stages of leaf andstem in plant growth (90d、1.5a、5a、10a、25a and50a). RACE technology was conducted todetect the cleavage sites in three predicted target genes. The major results are as the following:
(1) Small RNA library construction and Solexa sequencing for screening candidatemiRNA. Sequencing of sRNA was implemented using Illumina GAIIx platform. A total of27,042,027raw reads were obtained, and7,466,370clean mappable sequences were generatedafter filtering and removing contaminent, low quality, known other RNA sequences. Thesesequences were submitted to NCBI-Gene Expression Omnibus (GEO) database (GEO Number:GSE34805). Among them, a total of3,612,795sequences were21nt in length, approximatelyto48.39%of the total number of sRNA population, demonstrating that21nt sRNA in size wasthe main type. The query sequences were matched to the database of miRBase (17.0)(http://www.mirbase.org) and the larix transcriptome ESTs,308candidate miRNAs wereobtained. Solexa sequencing provided large amounts of candidate miRNA sequenceinformation for next miRNA identification.
(2) One hundred and seventy-four miRNAs from85miRNA families had been screenedusing BLAST and secondary structure prediction. There were77miRNAs from48conservedmiRNA families (47miRNAs were21nt in length,51miRNAs had uracil in which5′-endfirst base),97miRNAs from37novel families (72miRNAs were21nt in length,47miRNAshad uracil in which5′-end first base). Among them,20miRNA families were commonconserved in land plants;12miRNA families were conifer-specific. The expression detectionof34conserved and6novel miRNAs were validated by qRT-PCR. The knowledge of miRNAsequence characteristic in L.leptolepis will contribute to expand the miRNA information ofconifer trees.
(3) Two hundred and thirty-six mRNAs were predicted to be targets of85miRNAfamilies by bioinformatics. Target gene prediction and GO classification results indicated that85miRNAs targeted236target genes, which involved in all kinds of biological processesincluding transcriptional regulation, growth and development, protein transport, stressresponses, hormone signaling, electron transport and so on.
(4) The expression profiles of29miRNAs reduced during the early period of seedgermination,8miRNAs demonstrating a regular expression change pattern during the processof plant growth and development. The expression profiles of29miRNAs were analyzed by qRT-PCR during seed germination (0d,1d,5d,9d and28d) and plant growth (90d,1.5a,5a,10a,25a and50a) in larch. The results shown that all miRNAs were highly expressed in dryseed stage (0d) than subsequent stages, and the expression levels of13miRNAs (44.83%) allwent down gradually as a whole along with the progression of seed germination. The relativeexpression level of miR894was the highest, while the one of miR408was the lowest at acertain stage during seed germination. The expression levels of8miRNAs presented gradualupward or downward trends with increasing plant age. And5a and25a were the turning pointsof miRNAs differential expressions, which revealed that the two periods may be the criticalperiods of miRNA regulating the larch growth. The spatiotemporal and differentialexpressions of miRNAs may play important roles during seed germination and plant growth.
(5) The stabilities of12candidate internal reference genes were evaluated by geNorm andNormFinder softwar. The expressions of the tree candidate internal gene, APm、EF1and eIF,were most stable among different organs and developmental stages (somatic embryogenesis,seed germination and plant growth stage), providing optimum internal reference genes formiRNA target genes expression analysis using qRT-PCR.
(6) The predicted target genes of miR172, miR397and miR398were AP2L1, JR147962(Laccase) and JR143920(Phytocyanin) respectively. The target genes were found to becleaved most between10thand11thbase of complementary region of miRNAs and their targetgenes. The significant negative linear relationship between miR172and its target gene (AP2L1)was minus0.960（P=0.002).
In conclusion,174miRNAs from85families were identification, and8miRNAs mightbe involved in the regulation of growth and development of L.leptolepis. The significantnegative linear relationship between miR172and its target gene (AP2L1) was minus0.960.AP2L1is a crucial regulator being of great value for functional molecular studies duringconifer tree seed germination, growth and development. These results expand the number ofknown conifer miRNAs and provide a foundation for research on the expression patterns andfunctions of these miRNAs during individual development in conifer tree.
引文
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