鹽芥microRNA164基因功能研究

1、鹽芥microRNA164基因功能研究    鹽芥microRNA164基因功能研究 Research on Function of miR164 in Thellungiella Salsuginea【中文摘要】 microRNAs(miRNAs)是生物體內(nèi)源長(zhǎng)度約為20-23nt的非編碼小RNA,廣泛分布于真核細(xì)胞基因組中,是最大的基因家族之一,大約占到整個(gè)基因組的1%。在基因組上通常具有獨(dú)立的基因座位。1993年,Lee和Wightman等在研究線蟲(chóng)胚胎發(fā)育過(guò)程時(shí)發(fā)現(xiàn)了第一個(gè)mircoRNA基因lin-4。隨后,在擬南芥、水稻、玉米、高粱、甘蔗以及

2、苔蘚植物中先后發(fā)現(xiàn)了大量的miRNA。miRNA是一種負(fù)調(diào)控因子。在植物體內(nèi),初級(jí)轉(zhuǎn)錄產(chǎn)物在Dicer-like酶的作用下切割并自身折疊成不完全配對(duì)的發(fā)卡結(jié)構(gòu),然后參與組裝沉默復(fù)合體。通過(guò)與靶mRNA在剪切位點(diǎn)附近幾乎完全的互補(bǔ)配對(duì)而在轉(zhuǎn)錄后水平上對(duì)基因的表達(dá)進(jìn)行負(fù)調(diào)控,導(dǎo)致mRNA的降解或翻譯抑制,從而參與調(diào)控植物器官的形態(tài)建成、生長(zhǎng)發(fā)育、激素分泌、信號(hào)轉(zhuǎn)導(dǎo)以及對(duì)外界環(huán)境脅迫應(yīng)答等生物學(xué)過(guò)程。miR164由MIR164A、MIR164B、MIR164C三個(gè)基因座位編碼,靶基因?yàn)镹AC家族的六個(gè)轉(zhuǎn)錄因子。miR164通過(guò)調(diào)控這些靶基因影響生長(zhǎng)素梯度、側(cè)根的起始以及花瓣的數(shù)量。其中CUC1與CU

3、C2控制條分生組織的形成和器官邊界的發(fā)育。擬南芥中,miR164a缺失突變體和含有抗miR164的CUC2的突變體的葉片邊緣缺刻加深;相反,過(guò)量表達(dá)miR164的突變體葉邊緣平滑,過(guò)量表達(dá)抗miR164的CUC1也沒(méi)有出現(xiàn)此表型。CUC2失活造成miR164a缺失突變體或野生型葉緣缺刻消失。并且發(fā)現(xiàn)miR164a與CUC2的表達(dá)區(qū)域重疊,最初在幼葉的整個(gè)邊緣區(qū)表達(dá),然后逐漸局限于葉片邊緣凹進(jìn)的區(qū)域。因此,擬南芥中miR164通過(guò)負(fù)調(diào)控CUC2控制葉邊缺刻程度。2007年,JoseManuel Franco-Zorrilla等人在研究擬南芥磷饑餓誘導(dǎo)的miRNAmiR399時(shí)發(fā)現(xiàn)了一個(gè)非編碼蛋白

4、基因IPS1。IPS1含有與miR399互補(bǔ)配對(duì)的基序,但是這種互補(bǔ)配對(duì)被一個(gè)錯(cuò)配環(huán)打斷,錯(cuò)配環(huán)正好位于miR399的剪切位點(diǎn),導(dǎo)致IPS1不能被miR399剪切,反而螯合了miR399。并且這種結(jié)合比miR399與底物的結(jié)合更有競(jìng)爭(zhēng)性。因此,當(dāng)IPS1過(guò)量表達(dá)積累時(shí),大量的miR399與IPS1配對(duì),只有少數(shù)miR399與靶基因PHO2的mRNA配對(duì),發(fā)揮了剪切靶基因mRNA的作用,最終導(dǎo)致靶基因mRNA的大量積累,表現(xiàn)為枝條中磷含量的降低。這種miRNA抑制機(jī)制稱為靶基因模擬。靶基因模擬提供了一種新的分析miRNA功能的方法。鹽芥與擬南芥近緣,屬于十字花科植物。具有擬南芥很多同樣的優(yōu)點(diǎn)以作

5、為實(shí)驗(yàn)系統(tǒng):如類似的形態(tài)、小的基因組、短的生活史、豐富的種子和易于被轉(zhuǎn)化等。但是,鹽芥對(duì)多種脅迫的耐受能力遠(yuǎn)遠(yuǎn)高于擬南芥。是一種新型的鹽生模式植物。本實(shí)驗(yàn)以鹽芥為材料,設(shè)計(jì)兩對(duì)引物分別從擬南芥中克隆獲得MIR164基因和IPS1基因。一方面將MIR164基因連接本實(shí)驗(yàn)室設(shè)計(jì)的pCAMBIA-改造后的pCAMBIA 3301載體,構(gòu)建了MIR164過(guò)量表達(dá)載體,轉(zhuǎn)入農(nóng)桿菌浸染野生型鹽芥,獲得過(guò)量表達(dá)轉(zhuǎn)化株系。另一方面,將IPS1錯(cuò)配環(huán)兩端與miR399互補(bǔ)配對(duì)的片段替換成與miR164互補(bǔ)配對(duì)的片段,連接pCAMBIA-改造后的pCAMBIA 3301載體,構(gòu)建成MIR164沉默載體,轉(zhuǎn)入農(nóng)桿菌

6、浸染野生型鹽芥,獲得MIR164沉默轉(zhuǎn)化株系3株。同時(shí),為了驗(yàn)證鹽芥中CUC2也是miR164的靶基因,進(jìn)行了鹽芥CUC2同源基因的克隆。首先,提取鹽芥RNA,反轉(zhuǎn)錄獲得cDNA,然后設(shè)計(jì)一對(duì)簡(jiǎn)并引物,以鹽芥cDNA為模板進(jìn)行PCR,克隆得到鹽芥中CUC2同源基因長(zhǎng)268bp的片段,由于擬南芥中miR164與CUC2的結(jié)合位點(diǎn)位于774bp-794bp,因此又設(shè)計(jì)一個(gè)特異引物進(jìn)行3' -RACE?？寺〉玫紺UC2同源基因的3端序列。 【英文摘要】 MicroRNAs (miRNAs) are a class of endogenous noncoding single-stranded

7、 RNA with about 22-23 nucleotides length which are widely distributed in genome of Eukaryotic cells. microRNAs are one of the largest gene familise with the proportion of about 1% in genome which locad in independent locus. The first gene recognized to encode microRNA, lin-4 of C. elegans, were iden

8、tified on the basis of the developmental timing defects associated with loss-of-function mutations by Lee and Wightman in 1993. It is now known that there are hundreds of microRNAs genes in arabidopsis ,Oryza Sativa, Zea mays, Sorghum bicolo, Saccharum officenarum L and bryophyte. In plants, these n

9、ucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNA. microRNAs function as sequence-specific negative regulators in post-transcriptional gene silenci

10、ng by almost perfect complementarity with target mRNA around the cleavage site , which leads to mRNA cleavage or translational repression.By this way,miRNA function in Biological process,such as morphogenesis,growth and development,hormone secretion, signal transduction and the response capacity to

11、the external environmental stress.miR164 is encoded by MIR164A, MIR164B, and MIR164 C,whose targets are six members of the NAC family of transcription factors.miR164 function in auxin Auxin gradients ,the oritation of the Lateral root and the quantity of petal. CUC2 and CUC1 are required for embryon

12、ic shoot meristem formation and specification of the organ boundary.which are specificly regulated by miR164a. The mutations in the MIR164A gene deepen serration of the leaf margin. By contrast, leaves of plants overexpressing miR164 have smooth margins. Enhanced leaf serration was observed followin

13、g the expression of an miR164-resistant CUC2 but not of an miR164-resistant CUC1. Furthermore, CUC2 inactivation abolished serration in mir164a mutants and the wild type. CUC2 and MIR164A are transcribed in overlapping domains at the margins of young leaf primordia, with transcription gradually rest

14、ricted to the sinus, where the leaf margins become serrated. CUC2 specifically controls leaf margin development under the regulation of miR164.IPS1(INDUCED BY PHOSPHATE STARVATION1) is a nonprotein coding gene which is dicovered in the research of miR399 by JoseManuel Franco-Zorrilla1 et al. in 2007

15、.It contains a motif with sequence complementarity to the phosphate (Pi) starvationinduced miRNA-miR-399.The pairing is interrupted by a mismatched loop at the expected miR399 cleavage site. IPS1 mRNA is not cleaved but instead sequesters miR-399.IPS1 is a more efficient competitor for miR-399 compa

16、red with a cleavable substrate Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. We coin the termtarget mimicryto define this mechanism of inhibition of miRNA activity. Target mimicry provide a new method for

17、miRNA research。Thellungiella salsuginea is close relative to Arabidopsis which is also belong to Cruciferae and has good genetic features such as similar morphology, small genome size, short life cycle, high seed number and an efficient transformation method. However, T. salsuginea is able to withst

18、and dramatic salinity shock.We cloned IPS1 and miR164 form the genome of Arabidopsis.On one hand,we completed the construction of silencing vector with miR164,on the other hand,we constructed overexpressing vector by partly replacing the sequence of IPS1 with the sequence which is complementary to m

19、iR164.In order to check whether CUC2 is also the target gene of miR164 in T. salsuginea,we cloned the CUC2 Homologous gene in T. salsuginea by using Degenerate PCR and 3' -race. 【中文關(guān)鍵詞】 miRNA; miR164; IPS1; 缺刻; 鹽芥 【英文關(guān)鍵詞】 miRNA; miR164; IPS1; serration; Thellungiella salsuginea 【畢業(yè)論文目錄】摘要 7-9 AB

20、STRACT 9-10 第一部分 文獻(xiàn)綜述 11-26     1 MIRNA 簡(jiǎn)介 11-19         1.1 MIRNA 的發(fā)現(xiàn) 12         1.2 MIRNA 的生物合成及組裝 12-14             1.2.1 miRNA 前體的轉(zhuǎn)錄 12    &#

21、160;        1.2.2 miRNA 的加工和外運(yùn) 12-13             1.2.3 miRNA 組裝入沉默復(fù)合體 13-14         1.3 MIRNA 的作用機(jī)制 14         1.4 MIRNA 的功能 14-15  

22、60;      1.5 MIRNA 的鑒定 15-16         1.6 植物中保守的MIRNA 16         1.7 植物中不保守的MIRNA 16-17         1.8 MIRNA 與SIRNA 的比較 17         1.9 植物MI

23、RNA 靶基因的鑒定 17-19             1.9.1 植物miRNA 靶基因的鑒定 17-18             1.9.2 植物miRNA 調(diào)控范圍 18-19             1.9.3 植物miRNA 靶基因的實(shí)驗(yàn)確定 19 

24、0;   2 MIR164 與植物缺刻 19-22         2.1 植物葉片的發(fā)育及影響因子 19-20         2.2 MIR164 與CUC2 的突變體 20-22             2.2.1 miR164a 突變體缺刻加深 20-21      &#

25、160;      2.2.2 miR164a 靶基因的確定 21             2.2.3 CUC2 與MIR164A 的表達(dá)定位 21-22             2.2.4 葉片缺刻形成的模型 22     3 IP51 的發(fā)現(xiàn)及作用機(jī)制 22-23   

26、60;     3.1 IP51 的研究 22         3.2 IP51 的作用機(jī)制 22-23     4 排水器的結(jié)構(gòu)與發(fā)育 23-24     5 簡(jiǎn)并PCR 技術(shù) 24-25         5.1 簡(jiǎn)并引物的設(shè)計(jì) 24         5.2 簡(jiǎn)并PCR 技術(shù)的局限性 2

27、4-25         5.3 使用簡(jiǎn)并PCR 獲得基因全長(zhǎng)的方法 25     6 耐鹽模式植物鹽芥 25-26 第二部分 實(shí)驗(yàn)論文 26-48     1 實(shí)驗(yàn)材料 26-28         1.1 植物材料 26         1.2 菌種及質(zhì)粒 26      &#

28、160;  1.3 引物序列 26-27         1.4 酶及試劑盒 27         1.5 主要儀器 27         1.6 培養(yǎng)基及化學(xué)試劑 27-28         1.7 分析軟件 28     2 實(shí)驗(yàn)方法 28-35   &#

29、160;     2.1 MIR164 沉默載體的構(gòu)建 28-32             2.1.1 CTAB 法提取植物基因組DNA 28-29             2.1.2 克隆IP51 序列 29           

30、0; 2.1.3 Gene Clean 方法從瓊脂糖凝膠上回收DNA 片段 29             2.1.4 改造的與miR164 互補(bǔ)的IP51 序列 29-30             2.1.5 改造的IP51 連接載體 30-31             2.1.6 大腸桿菌感受態(tài)的制備及轉(zhuǎn)化 31             2.1.7 質(zhì)粒的提取 31-32             2.1.8 酶切驗(yàn)證 32