糯稻種原之蜡質基因DNA 序列與胚乳澱粉組成之相關性研究

Abstract

Studies on the correlations between variation in waxy gene sequences and starch components in endosperm for glutinous-rice germplasm

本試驗使用144 個稻種原(包括118 個糯性種原與26 個非糯性種原)做為材料，目的為研 究蠟質基因在糯稻背景下，偵測Wxa allele 是否在梗稻或糯稻中出現，或者蠟質基因序列上的 差異使否會影響該基因的作用。因為米粒外觀與直鏈澱粉在判別是否為糯稻種原的標準上， 存在些許差異，造成10 個由外觀觀察歸類為糯性的種原，卻具有高直鏈澱粉含量。在轉譯起 始點下游144 bp 處，核甘酸 ”G” 表示Wxa 對偶基因，而 ”T” 則表示Wxb 對偶基因，在所有 參試種原中，此處的差異可解釋42.6 %的直鏈澱粉變異。此外，在8 個(CT)重複序列基因座 中，6 個對直鏈澱粉具有顯著的作用，其中(CT)11,(CT)17 與(CT)10 各別對直鏈澱粉變異的貢獻 為34.5 %，19.5 %及18.7 %。在本實驗中，也另外發現(AATT)6 與另外三個鹼基置換點(第229、 363、390 bp 處)對於直鏈澱粉有顯著貢獻。另外，我們也發現有3 個糯性種原(C102, C105, and C124)與4 個梗稻種原(C010, C050, C093, and C100)具有Wxa 對偶基因，此結果推翻先前所認 知的結果：糯稻及梗稻僅具有Wxb 對偶基因。同時，本試驗由蠟質基因的序列訊息，建立了 該基因的樹狀圖; 該144 個種原的分布，主要依據(CT)重複序列、直鏈性澱粉含量、及引種 國家進行分群，結果與先前ISSR 分子標誌針對秈糯及梗糯分群結果相似。設計引子偵測Wx 基因長度結果發現種原間並無明顯差異。由GLM 分析結果，測得144-G/T 變異及(CT)重複序 列與蠟質基因表現有關，另外，亦新測得(AATT)5/6、390-C/T、363-T/G 及229-G/A 影響蠟質 基因表現。其中(AATT)5/6、390-C/T、363-T/G 可解釋40.2 % 的直鏈澱粉變異，而229-G/A 則可貢獻54.9 % 。

To study waxy gene expression under glutinous background, to detect Wxa allele in glutinous or japonica rice (Oryza sativa L.) varieties, to identify sequences related waxy gene expression, and to evaluate genetic similarity among glutinous rices, the 5' upstream sequences of waxy alleles among 144 rice germplasm, (including 118 waxy and 26 non-waxy), were analyzed. Because ten germplasm classified as waxy-type by their appearance had higher amylose amount (>12%), there was a little difference in judging waxy-type varieties between by checking grain appearance and amylose content. The diverse nucleotides “G” and “T” respectively representing Wxa and Wxb were found in glutinous germplasm. The single nucleotide substitution could explain 42.6 % of amylose content. Besides, we have found eight alleles of (CT) repeat in our study, and six of the eight alleles have significant effects on amylose contents. The alleles (CT)11, (CT)17 and (CT)10 contributed 34.5 %, 19.5 % and 18.7 % of total amylose variation, respectively. The finding of three glutinous (C102, C105, and C124) and four (C010, C050, C093, and C100) japonica germplasm carrying a Wxa allele has refreshed the previous understanding that glutinous or japonica rice carry Wxb allele only. According to the sequence of waxy alleles, the gene tree of 144 germplasm was established. The investigated varieties were separated predominantly by repeat numbers of (CT), amylose content, and origins of germplasm. Similar results have been found in clustering based on ISSR markers previously. From the GLM analysis results, 144th(G/T) and (CT) repeated alleles were detected and other newly discovered factors including (AATT)5/6, 390-C/T, 363-T/G, and 229-G/A. have a significant attributions for amylose accumulation. (AATT)5/6, 390-C/T, and 363-T/G could explain 40.2 % of amylose variantion, and 229-G/A could contribute 54.9 %.