水稻T-DNA 插入突變點兩側序列解序方法之比較及插入點之研究

The study of flanking sequences generate methods and the insertion sites of rice T-DNA mutant lines

鄭堯中(chêng yao chung)

指導教授 ： 范宗宸

亞洲大學/人文社會學院/生活應用科學學系碩士班/碩士(2007年)

摘要

目前從事水稻功能性基因之研究主要以 T-DNA 作為 Tag 以利進一步研究，對於逆向遺傳學的研究來說，獲得 T-DNA 插入突變點之(Flanking sequence tag, FST) 是不可或缺的，我們可藉由所獲得的 FST基因型，與植株表現型相比對，進而研究瞭解改變表現型突變之基因為何，及可能的機制以導致此種性狀之表達。因此，建立 T-DNA 插入突變點之 FST 資料庫，可加速水稻功能性基因體的研究。本研究希望藉由一個新開發出的 PCR 方法: Hooker adaptor PCR，來快速獲得FST 的資訊。
本研究繁殖的 1301 個品系中，總共有 319 個品系有明顯的外觀變異。植物 DNA 利用 CTAB 法萃取，再利用特定的限制酵素切割後，藉由 PCR 方法進行 T-DNA 插入點之 FST 解序。結果得到總共 885條帶，其中以 Inverse PCR (IPCR) 獲得 139 條條帶，AL-PCR獲得 382條條帶，HA-PCR獲得 364 條條帶。
IPCR 解序以 M48821-4、M66317 兩株突變株為例，比對 TRIM 結果， M48821-4之T-DNA 插入水稻第四條染色體， M66317 T-DNA 插入第九條染色體。AL-PCR 以 M27669-8 突變株之 FST 為例，成功解序出 RB 及 LB 之序列，其插入位點幾乎相同且非常相近，皆位於水稻第八條染色體。本研究利用HA-PCR成功解序M60023、M60035 ( 2 x copy ) 兩株突變株之 FST，比對 TRIM 結果 M60023 插入水稻第八條染色體，M60035獲得兩條不同 T-DNA 及 FST，插入位點分別為第六及第九條染色體。
於461 株水稻台農67 號 T-DNA 插入染色體之分佈研究結果顯示，其T-DNA 主要分佈在第1、2、3 及第6 條染色體，第9 與第12 條染色體的分佈則為最少的。
以IPCR、AL-PCR 及HA-PCR 解序效能比較的結果顯示，IPCR 成功率為17%，得到一個品系條帶時間為3 天。AL-PCR 成功率為21%，得到一個品系條帶時間為3 天。HA-PCR 成功率為56%，得到一個品系條帶時間為1 天。比較後發現 HA-PCR 不但操作簡單，成本的消耗及時間的花費都比其他兩者來的少，HA-PCR 對目前現階段的解序技術來說是非常重要且新穎的，我們認為此為水稻T-DNA 突變品系兩側序列解序之最佳方法。

並列摘要

The researches engaged in the functional genomic of rice at present mainly used T-DNA as tag to understand the profits of rice gene and there function. In addition, for the purpose of reverse genetic, the research from gene sequence to the phenotype by T-DNA insertion the inseration sites flanking sequence is very importment.Which can let us understands the connection between phenotypes and their gene sequences of those mutane lines. The database of T-DNA insertion sites (Flanking sequence tag, FST) which can accelerate the research of the rice functional genomic, become an importment issue.
In this research, we intend by develop a newly PCR method which was named as Hooker adaptor PCR to solute this problem. Totally 1301 lines were planted in the quarantine green house in Taiwan Agricultural Research Institute in this research. Among them 319 lines have obvious appearances different in phenotype.
The CTAB method was used to extract plant DNA, after the genomic DNA was extract, the restriction enzyme and ligase was used, on the T-DNA insertion sites flanking sequence was generated by PCR method. The results shows, totally 885 bands was generated, among them 139 bands by Inverse PCR (IPCR),382 bands by adaptor ligation PCR(AL-PCR),and 364 bands by hooker adaptor PCR (HA-PCR).
Those FST generates by IPCR, was showed by rice T-DNA mutant lines M48821-4 and M66317 as examples. After compare with TRIM, the results showed that the T-DNA was inserted in the Chr 4 and Chr 9 of rice respectively. We take M27669-8 as an example for those T-DNA inserted mutant lines their FST were generated by AL-PCR.The flanking sequences of RB and LB compared with TRIM BLAST, the result showed the RB and LB sequence were perfect inserted in the orderly closed location,which is located in rice Chr 8. As for Hooker adaptor PCR (HA-PCR) not only generated 364 bands but also succeed in solving the problem of FST which can not generated by IPCR or AD-PCR. We demonstrated by M60023 and M 60035 (2 copy) flanking sequences after blast in TRIM. The results show ed that M60023 have an insert in Chr 8. M60035 have two different T-DNA inserted which located at Chr 6 and Chr 9.From the results of 461 lines of TN67 T-DNA insertion mutants, the distribution of the T-DNA is mainly in the Chr 1, Chr 2, Chr 3 and the Chr 6. The Chr 9 and Chr 12 have the lease distribution.After analysis the cost and successed ratio, it showed that the success rate of IPCR is 17%,it take 3 days to get a line’s FST. Success rate for AL-PCR is 21%,it takes 3 days to get a line’s FST.The successful rate for HA-PCR was 56% it take one day to get a line’s FST.The result shows the HA-PCR not only easy to operate, lest cost and lease time consumption compare to the others. The HA-PCR is very efficient and novel technology of perfect at the present stage. We believe this method can be thought as the best method for generated T-DNA insertion sites flanking sequence of rice.

並列關鍵字

T-DNA

參考文獻

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