本文選題：黃藤 + NAC　； 參考：《林業(yè)科學(xué)》2017年08期

【摘要】：【目的】棕櫚藤是重要的森林植物,纖鞭是其重要的攀援器官,也是重要的分類依據(jù)。研究黃藤NAC(NAM,ATAF和CUC)轉(zhuǎn)錄因子基因的分子特征并開發(fā)SSR分子標記,以期為棕櫚藤的分子育種和輔助分類提供參考依據(jù)�！痉椒ā恳渣S藤為材料,借助轉(zhuǎn)錄組數(shù)據(jù),采用同源克隆的方法從黃藤中分離NAC基因,采用生物信息學(xué)方法進行基因結(jié)構(gòu)、蛋白性質(zhì)與結(jié)構(gòu)分析和SSR位點預(yù)測,采用實時定量PCR技術(shù)分析基因的組織表達特性,利用PAGE電泳和測序技術(shù)分析SSR分子標記在不同棕櫚藤樣品中的通用性和多態(tài)性�！窘Y(jié)果】從黃藤葉片中獲得了2個NAC同源基因DjNAC3(Gen Bank登錄號:KU556738)和DjNAC4(Gen Bank登錄號:KX579750),二者的開放閱讀框長度分別為729 bp和1 326 bp,對應(yīng)的基因組序列為850 bp和1 441 bp,均包含2個外顯子和1個內(nèi)含子。DjNAC3和DjNAC4編碼的蛋白分別為242 aa和441 aa。蛋白結(jié)構(gòu)分析表明,DjNAC3和DjNAC4具有典型的NAC轉(zhuǎn)錄因子結(jié)構(gòu)特征,屬于NAC家族的CUC亞家族,但二者之間的相似系數(shù)僅為23.6%,表明它們在黃藤生長發(fā)育過程中可能具有不同的功能。DjNAC3和DjNAC4在不同組織中的表達模式存在明顯差異,DjNAC3在發(fā)育成熟纖鞭中的表達豐度最高,葉片中的表達豐度最低,而DjNAC4則在發(fā)育成熟的鉤刺中表達豐度最高,而發(fā)育初期的鉤刺中最低。在DjNAC3和DjNAC4的基因組序列中分別包含1個SSR位點,其中前者的SSR位點位于內(nèi)含子區(qū)域,為(TA)6,后者的SSR位點位于第1個外顯子區(qū)域,為(GCA)5。根據(jù)DjNAC3和DjNAC4中SSR位點旁側(cè)序列設(shè)計引物,以黃藤和另外20個不同棕櫚藤樣品的基因組DNA為模板進行擴增,PAGE電泳結(jié)果分析表明,引物具有較高的通用性,且擴增產(chǎn)物具有多態(tài)性。6個樣品的測序結(jié)果證實,用DjNAC3設(shè)計引物的擴增產(chǎn)物測序獲得的SSR位點序列存在著一定的差異,既包括SSR類型變異、重復(fù)次數(shù)變化等多態(tài)性,又有SSR位點缺失的現(xiàn)象;而根據(jù)DjNAC4設(shè)計引物擴增獲得的SSR位點序列差異主要為重復(fù)次數(shù)的變化�！窘Y(jié)論】黃藤DjNAC3和DjNAC4基因的基因結(jié)構(gòu)、表達模式、SSR位點等均存在明顯的差異,這表明它們在黃藤生長發(fā)育中可能具有不同的功能,二者所包含的SSR分子標記具有通用性和多態(tài)性,可以作為分子標記應(yīng)用于棕櫚藤的輔助分類和分子輔助育種。
[Abstract]:[objective] Rattan is an important forest plant, the whip is an important climbing organ and an important taxonomic basis. To study the molecular characteristics and develop SSR molecular markers of transcription factor gene of NAC (NAMU ATAF and CUC), in order to provide reference for molecular breeding and auxiliary classification of rattan. [methods] with the aid of transcriptome data, NAC gene was isolated by homologous cloning method. The gene structure, protein properties and structure analysis and SSR site prediction were analyzed by bioinformatics. The tissue expression characteristics of NAC gene were analyzed by real-time quantitative PCR. Page electrophoresis and sequencing techniques were used to analyze the versatility and polymorphism of SSR markers in different samples. [results] two NAC homologous genes DjNAC3 (Gen Bank accession number: KU556738) and DjNAC4 (Gen Bank accession number: KX579750) were obtained from leaves of Rhizoma lucifera. The length of the open reading frame was 729 BP and 1 326 BP, respectively, and the corresponding genomic sequence was 850 BP and 1 441 BP, respectively, containing two exons and one intron. DjNAC3 and DjNAC4 encoded a protein of 242aa and 441aa, respectively. The protein structure analysis showed that DjNAC3 and DjNAC4 had typical structural characteristics of NAC transcription factors and belonged to the CUC subfamily of NAC family. But the similarity coefficient between them was only 23.6B, which indicated that they might have different functions during the growth and development of Rhizoma vines. There were obvious differences in the expression patterns of DjNAC3 and DjNAC4 in different tissues. The expression of DjNAC3 was the most abundant in the mature whip. The expression abundance of DjNAC4 was the lowest in the leaves, but the highest in the mature and the lowest in the early stage of development. One SSR locus was found in the genomic sequence of DjNAC3 and one SSR locus was found in the intron region of DjNAC4. The SSR locus of the latter was (TA) 6, and the SSR locus of the latter was (GCA) 5. The primers were designed according to the SSR sequences in DjNAC3 and DjNAC4, and the genomic DNA of Rhizoma sinensis and 20 other samples were used as templates. The results of page electrophoresis showed that the primers had high versatility. The sequencing results of 6 samples confirmed that there were some differences in the sequence of SSR loci obtained by PCR products sequenced with DjNAC3 primers, including SSR type variation, repeat frequency variation and so on. The sequence difference of SSR locus based on DjNAC4 primer amplification was mainly the variation of repeat times. [conclusion] there were significant differences in gene structure, expression pattern and SSR loci of DjNAC3 and DjNAC4 genes. These results suggest that they may have different functions in the growth and development of Rattan, and the SSR molecular markers contained in them are versatile and polymorphic, which can be used as molecular markers for auxiliary classification and molecular assisted breeding of Rattan.
【作者單位】： 國際竹藤中心國家林業(yè)局竹藤科學(xué)與技術(shù)重點開放實驗室;河北農(nóng)業(yè)大學(xué)園藝學(xué)院;
【基金】：“十二五”農(nóng)村領(lǐng)域國家科技計劃項目“竹藤種質(zhì)資源創(chuàng)新利用研究”第一課題“竹藤優(yōu)異種質(zhì)創(chuàng)制創(chuàng)新與種苗培育標準化示范”(2015BAD04B01)
【分類號】：S792.91

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