【摘要】：【目的】基于前期鵝掌楸生長性狀與EST-SSR分子標記關聯分析結果,克隆分離與鵝掌楸生長性狀相關聯的SSR位點相對應的基因,通過分析其序列特征、結構特征、與其他物種同源基因的親緣關系和表達特性,初步了解該基因在鵝掌楸生長發育過程中的作用。同時,利用遺傳轉化技術對分離的基因進行功能研究,以期為鵝掌楸生長發育的分子機制研究及功能基因挖掘奠定基礎。【方法】以鵝掌楸葉芽為材料,借助其葉片轉錄組數據庫,采用RT-PCR和RACE技術克隆分離與鵝掌楸生長性狀相關聯的642號位點對應的EST序列相關基因,對其進行生物信息學分析。通過實時熒光定量PCR檢測該基因在鵝掌楸花芽、盛花期的葉芽、葉片、花瓣、雄蕊、雌蕊中的相對表達量。利用Gateway技術構建該基因的植物過表達載體,使用農桿菌GV3101介導的花絮浸染法轉化擬南芥,獲得T2代轉基因植株,直接觀察轉基因植株表型。【結果】克隆獲得與鵝掌楸生長性狀相關聯的642號位點相對應的基因,該基因全長1 968 bp(GenBank登錄號:KU883608),開放閱讀框為1 269 bp,編碼422個氨基酸。生物信息學分析表明該基因編碼的蛋白序列含有1個典型的DHHC-CRD結構域,屬于DHHC型鋅指蛋白家族成員,與其他植物預測的蛋白質棕櫚酰基轉移酶(PAT)高度相似,并根據與NCBI中擬南芥PAT基因的比對結果,將其命名為LcPAT8。組織表達分析表明,LcPAT8基因在鵝掌楸花芽、葉芽等6個組織中均有表達,在雌蕊中表達量最高,花瓣和葉片中的表達量高于葉芽和花芽,而在雄蕊中的表達量最低。利用Gateway技術,成功構建了鵝掌楸LcPAT8基因的植物過量表達載體,獲得T2代轉基因擬南芥。與野生型擬南芥相比,過表達LcPAT8基因的擬南芥植株的蓮座葉片數目以及抽薹時間沒有發生明顯變化,而野生型植株大部分角果成熟、葉片枯黃掉落時,轉基因植株依然生長旺盛,葉片鮮綠并且還有大量花絮,在野生型植株干枯死亡后,轉基因植株還有大量側枝生長、開花。【結論】鵝掌楸LcPAT8基因在雌蕊中表達量最高,其次是花瓣,可能參與花瓣的擴展、心皮及胚的發育;在擬南芥中過表達LcPAT8基因,明顯延長了植株的生長期。因此,鵝掌楸LcPAT8基因可能在植物生長發育過程中起著重要的調控作用。
[Abstract]:[objective] based on the results of early correlation analysis of growth traits and EST-SSR markers in Liriodendron chinense, the genes corresponding to SSR loci associated with growth traits of Liriodendron were cloned and isolated, and their sequence and structural characteristics were analyzed. The relationship and expression characteristics of the homologous gene with other species were studied to understand the role of the gene in the growth and development of Liriodendron. At the same time, the function of the isolated genes was studied by genetic transformation technique, in order to lay a foundation for the study of molecular mechanism and functional gene mining of Liriodendron chinense. [methods] the leaf buds of Liriodendron were used as materials. With the help of the leaf transcriptome database, RT-PCR and RACE techniques were used to clone and isolate the EST sequence associated with the growth traits of Liriodendron (Liriodendron mandshurica) at locus 642, and its bioinformatics analysis was carried out. The relative expression of the gene in flower bud, leaf, petal, stamen and pistil of Liriodendron was detected by real-time fluorescence quantitative PCR. The plant overexpression vector of this gene was constructed by Gateway technique, and transformed into Arabidopsis thaliana by Agrobacterium GV3101 mediated floss soaking, and T2 generation transgenic plants were obtained. The phenotype of transgenic plants was observed directly. [results] A gene corresponding to locus 642 associated with growth traits of Liriodendron chinense was obtained. The gene was 1 968 bp (GenBank accession number: KU883608) and the open reading frame was 1 269 bp,. Encode 422 amino acids. Bioinformatics analysis showed that the protein sequence encoded by the gene contained a typical DHHC-CRD domain and belonged to the DHHC zinc finger protein family, which was highly similar to the predicted protein palmityl transferase (PAT) from other plants. According to the comparison results of Arabidopsis thaliana PAT gene with NCBI, it was named LcPAT8.. Tissue expression analysis showed that LcPAT8 gene was expressed in flower bud and leaf bud of Liriodendron, and the highest expression was in pistil. The expression of LcPAT8 gene in petals and leaves was higher than that in leaf bud and flower bud, but lowest in stamen. The plant overexpression vector of LcPAT8 gene of Liriodendron was constructed by Gateway technique, and T2 generation transgenic Arabidopsis thaliana was obtained. Compared with wild-type Arabidopsis thaliana, the number of rosette leaves and bolting time of Arabidopsis thaliana plants with overexpression of LcPAT8 gene did not change significantly. The transgenic plants still grow vigorously, leaves are fresh green and there are a lot of flowers. After the wild-type plants withered and died, the transgenic plants still have a large number of lateral branches growing and flowering. [conclusion] the LcPAT8 gene of Liriodendron has the highest expression in pistil. The second is petal, which may be involved in the expansion of petals and the development of carpels and embryos. Overexpression of LcPAT8 gene in Arabidopsis thaliana significantly prolonged the plant growth period. Therefore, the LcPAT8 gene of Liriodendron mandshurica may play an important role in plant growth and development.
【作者單位】： 南京林業大學南方現代林業協同創新中心;貴州省林業科學研究院;
【基金】：國家自然科學基金項目(31470660) 江蘇省高校優勢學科(PAPD)
【分類號】：S792.21

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