表現輔助蛋白提昇農桿菌之水稻轉殖效率與水稻突變株rolts之性狀分析

Abstract

農桿菌常被廣泛使用於植物基因轉殖技術，因其操作簡易，可使目標基因單一且完整的插入染色體組，而不易引發基因靜默(gene silencing)反應。近30年來以此法進行轉殖且成功的案例不少，但仍舊有許多植物或品種難以進行轉殖(recalcitrant plant)，其中包括蘭花、百合等重要花卉作物，及大豆、玉米、秈稻等重要經濟作物也是如此，因此提高植物轉殖效率仍舊是極為重要的課題。農桿菌基因轉殖步驟主要是藉由T-DNA傳送至植物中，而T-DNA的傳送過程需要許多輔助蛋白(accessory protein)參與，許多文獻指出，大量表現某些輔助蛋白的基因轉殖擬南芥，其下一個世代植株之轉殖效率大幅提升；故本實驗欲藉由暫時性大量表現輔助蛋白，提升轉殖當代目標基因之轉殖效率。以擬南芥輔助蛋白序列為藍本，選殖水稻或農桿菌的同功基因(gene ortholog)，稱之為促轉基因（Enhance Transformation genes，簡稱為ET gene）。本研究以水稻為測試對象，使用一個帶有促轉基因質體的農桿菌，及另一個帶有GUS報導基因質體的農桿菌共同感染植物。藉由暫時性與永久性轉殖分析評估這些促轉基因之功效，將促轉基因與空載體相比，BTI1、VirE3、VIP1(9)與H2A之轉殖效率可提升1.2~2.2倍。農桿菌VirB2交互蛋白（BTI1）為最理想且有效之促轉基因，其可以促進目標基因之暫時性與永久性轉殖效率，且不會異常提升目標基因拷貝數，自行嵌入染色體組的頻率也不會特別上升。在大量表現BTI1 之轉殖株中，WAKY54與WAKY6之表現量顯著降低，導致農桿菌感染能力上升。由於BTI1屬於Reticulon-like proteins，其可能與參與植物免疫反應之FLS2接受器有交互作用，因此降低植物免疫反應，進而增進農桿菌之感染能力。

TNG67與SA1613.1均為正常形態且豐產的水稻品種(系)，但其F2雜交子代在分蘗數及根性狀上呈現連續分布，暗示該性狀係屬數量遺傳調控。利用F2族群定位目標基因座，得知分別於第1號與第11號染色體上各存有一個候選基因座，分別命名為q1PN與q11PN。兩基因座單獨影響外表型變異值(variation of phenotype)皆高達38.5%，為主效基因座，而其交感作用亦顯著影響外表型變異值達12%，故q1PN與q11PN調控水稻穗數性狀變異值共計為89%。由於q1PN十分靠近中節（centromere），且附近有多個物理間隙（physical gap）存在，故決定將重心放在q11PN上，並將此定名為rolts基因座。各品系基因型以逗號前後分別代表q1PN與q11PN之基因型，當基因型組合為[T,S] (T：TNG67同型接合基因型，S：SA1316.1同型接合基因型，H：heterozygous異型接合基因型) 時，植株外表型為弱小單分蘗，且幾乎無根，稱此性狀為rolts (rootless plant with tiny single tiller)。使用水耕栽培針對rolts性狀進行觀察，結果顯示基因型是[T,S]的植株在種子萌芽5天後，就出現種子根較短及不定根數降低的現象。雖然其節點(node)與不定根根源體與正常植株並無差異，表示應是根部結構異常所致。利用冷凍掃描式電子顯微鏡觀察14天秧苗的種子根，發現成熟區橫向結構並無異常；以共軛焦顯微鏡觀察，發現種子根縱向組織於長度出現劇烈變化，細胞延長區長度明顯變短，且每個表皮細胞的長度也較短，顯示rolts 植株之細胞無法延長。利用次世代定序分析（RNA Seq）顯示於rolts基因與澱粉代謝有高度相關，暗示其可能參與細胞壁生合成或營養攝取等作用，直接或間接影響細胞延長。並根據精確定位分析，將候選基因座限縮至1.66 Mb。

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. The method results in mostly single or low-copy integration of full-length T-DNA, and is less likely to trigger gene silencing than gene-gun mediated method. Various technical modifications, either on Agrobacterium or plant side, were employed to improve the transformation efficiency ever since its invention on 1983. However, many plant species or cultivars are still granted as “recalcitrant” that barely give rise to regenerable and non-chimeric transgenic lines. Thus, transformation technology remains a challenging issue to be resolved. The long journey for T-DNA transferring in plant transformation not only relies on Agrobacteria proteins but also is aided by various plant factors. Interestingly, progenies of transgenic Arabidopsis overproducing several accessory proteins were found to be hypersensitive in further transformation. In this study, we aim to investigate that can transformation efficiency be enhanced by temporarily co-overproducing accessory proteins while transformation of target gene. Using Arabidopsis sequence as template, orthologous genes encoding the accessory proteins were isolated from rice and named as ET gene for their putative roles in “enhancing transformation”. Co-transformation of two Agrobacteria, with one deliver ET gene and the other provide target gene together with antibiotics selection, were tested on rice. Screened by transient transformation assays and confirmed by permanent transformations, four genes including BTI1, VIP1(9), VirE3, and H2A, were found to confer 1.2~2.2 fold higher transformation efficiency than the vector control. Transgenic lines regenerated from the above co-transformation processes were evaluated for the transformation frequency, the ET-gene co-integration frequency, and the copy number of target gene. BTI1 that possess the VirB2-interacting activities was found to exhibit ideal characteristics expected for an ET gene. In the BTI1-overexpressing rice line, increase of transcript amount of OsWRKY54 and OsWRKY6 in responding to Agrobacterium infection was rather limited. As BTI1 is also known as the Reticulon-Like Proteins required for the subcellular targeting of FLS2, this result suggests that the plant PAMP-triggered immunity, act via the FLS2-depenednt pathway, may be compromised in the BTI1-overepxressing rice callus and therefore render plant with higher susceptibility to Agrobacteria infection.

TNG67 and SA1613.1 both are normal rice lines with high yield. However, their F2 progenies exhibited wide segregations in panicle number and root biomass. A continuous phynotypic distribution suggests quantitative trait loci (QTL) controls underneath. Primary mapping on a F2 population revealed two major QTLs, q1PN and q11PN, explained ~89% phenotypic variation including ~38.5% from each locus independently and ~12% from their interaction. As q1PN is very close to the centromere of chromosome 1 which may hamper it from fine-mapping, q11PN is chosen as the target for study. When both q1PN alleles are from TNG67, allele of q11PN from SA1613.1 acts semidominantly and generates rootless plant with tiny single tiller, abbreviated as “rolts”. Continued hydroponic culture of rolts significantally increase its tiller number but not the root formation, suggesting no major defects in the aerial organs of rolts. Compared with wild-type rice, rolts seedlings exhibited shorter seminal root and fewer adventitious root (crown root) early on 5 day old seedlings. Although the nodes and crown root promordia surrounding node remain similar to that of the wild-type rice, elongation of root seem to be sererely hampered, eventually cause a rootless phenotype. Dissections of young seminal root of rolts revealed that the lateral-patterning in the maturation zone is bascially normal, in contrast to a drastic decrease in length of the longitudinal elongation zone. As all epidermal cells at the maturation zone of young rolts seedlings remain very short, it is concluded that the rolts phenotype may mainly caused by defect in cell elongation. Revealed by transcriptome analysis, genes related to starch metabolism that involved in cell wall biosynthesis and/or nutrition supply were differentially expressed in rolts. As mapping experiments defined rolts genes within a 1660 kb region on chromosome 11, candidate rolts gene will be examined based on its putative roles involving cell elongation.