番木瓜MADS box E-class基因CpMADS1、 CpMADS3與TM6 系基因CpMADS2之選殖與分析

Abstract

番木瓜(Carica papaya L.)為世界重要果樹，2006年全球栽培面積約40萬公頃，其具雌株、雄株和兩性株三種性型植株，兩性株可自花授粉，子腔小運銷成本較低，果肉利用率較高，故大部分番木瓜生產國家以栽培兩性株為主。但兩性株花朵受環境等影響，易造成異常的花朵性型表現，產生畸型果或開花不結果，影響產量與經濟產值。 番木瓜花性型表現主要決定於花器形成過程，為瞭解花器決定MADS box基因於番木瓜花朵發育與性別決定所扮演之角色，本試驗以番木瓜花芽為材料，選殖了MADS box E-class基因CpMADS1、CpMADS3與屬於B-class TM6系基因CpMADS2。由RT-PCR 和real-time PCR 分析結果顯示CpMADS1和CpMADS3 於內三輪花器表現，以心皮表現量最大，雄蕊、花瓣次之，CpMADS2則表現於花瓣及雄蕊，顯現B-class和E-class MADS box基因之特性，且此三個花器分化基因在番木瓜花朵發育極早時期（花芽小於1mm）就大量表現。以此3個基因為探針，進行不同性別，包括雌株、雄株、兩性株和特殊之後裔全兩性、後裔全雄性共10個木瓜品系基因組DNA之南方雜合分析，結果偵測雜合之限制酶切片段及數目於植株性別及品系間無差異，顯示花器分化基因CpMADS與木瓜植株性別決定可能不是在基因構造層次。進一步進行E-class基因CpMADS1、CpMADS3轉殖於煙草試驗，經CpMADS1和CpMADS3基因全長以限制酶切及黏合反應轉入pCAMBIA1305.3雙偶載體，並以電穿孔法轉型於農桿菌LBA4404後，將基因轉殖於‘Wisconsin 38’煙草，轉殖培植體經由抗生素篩選、組織GUS活性之化學染色分析及PCR確認後，經馴化栽培至開花。CpMADS1和CpMADS3基因轉殖煙草植株外觀、花序、開花時間無明顯改變，細部觀察花朵的四輪花器也與野生型相同。

Papaya (Carica papaya L.) is an economically important fruit. The harvested area around the world is about 400 thousand hectares in 2006. Papaya plants are trioecious, with female, male and hermaphrodite, respectively. In hermaphrodite papaya, male flower and imperfect hermaphrodite flower are often observed because the floral organ development may be influenced by environmental and hormonal factors. Despite various causes led to malformation of papaya fruits, hermaphrodite papayas are favored worldwide for economic production. However, several drawbacks were observed during the formation of bisexual flowers, such as pistil degeneration and carpel-like anthers, which led to fruit malformation and resultant yield loss. Sex expression in papaya flowers probably is determined very early during floral differentiation. In order to understand the roles played by the MADS-box genes in flower development and sex determination, cDNAs of E-class genes CpMADS1, CpMADS3 and a TM6 lineage of the B-class gene CpMADS2 were cloned from young flower buds of papaya. RT-PCR and real-time PCR analyses revealed that CpMADS1 and CpMADS3 were preferentially expressed in the carpel and also expressed in petals and stamens. CpMADS2 was expressed in both petals and stamens. These genes all expressed early during floral development. Comparison of ten papaya genotypes with 5 different sex phenotypes, viz. hermaphrodite, male, female, progeny-all-hermaphrodite, progeny-all-male, by Southern blot analysis of the genomic DNAs using probes of the three genes revealed similar restriction pattern and copy number, suggesting low correlation at genomic level of the three CpMADS genes with sex expression of papaya plants. The CpMADS1 and CpMADS3 genes were constructed respectively in a binary vector and introduced into tobacco (Wisconsin 38) genome using Agrobacterium- mediated transformation. Amplification by polymerase chain reaction of genomic DNA confirmed the integration of the CpMADS1 and CpMADS3 genes into the transgenic tobacco genome. However, the phenotype of tansgenic tobacco plants carrying CpMADS1 or CpMADS3 were similar to the wild type.