摘要
本研究以海岛棉基因组为材料,从海岛棉Pima90-53 BAC文库中筛选棉属染色体组特异重复序列。通过荧光原位杂交验证筛选出来的重复序列在四倍体海岛棉,二倍体亚洲棉、草棉、阿非利加棉、雷蒙德氏棉染色体上的分布情况；将筛选出来的BAC重复序列构建亚克隆,对其亚克隆的序列进行了分析。主要研究结果如下：
1.从Pima90-53 BAC文库中共筛选了19,200个克隆,以海岛棉有丝分裂中期染色体为靶DNA,将通过菌落原位杂交筛选出来的信号较强的BAC单克隆作为探针进行荧光原位杂交。根据荧光信号在海岛棉染色体上的分布情况来看,筛选出来的BAC单克隆共有4种,其克隆编号分别为：428K20、412A11、334E15、216B18。
2.原位杂交结果表明：428K20在海岛棉所有染色体上均产生了杂交信号,其中在较大染色体(A亚组)上除染色体两端外其余部分均产生了强烈的杂交信号,而较小染色体(D亚组)上仅在着丝粒区域产生明显的杂交信号,428K20在亚洲棉所有染色体上除染色体两端其余部分均产生了很强的杂交信号,但其在雷蒙德氏棉上却没有产生信号；412A11只在海岛棉较大染色体(A亚组)两端产生杂交信号,在亚洲棉、草棉、阿非利加棉所有染色体两端均产生了明显的杂交信号,与428K20一样在雷蒙德氏棉上没有信号产生,因此可证实428k20与412A11均由A基因组特异的重复序列组成,但428K20还含有一种可转座的因子(序列)。Pima90-53有丝分裂中期染色体以334E15作为探针进行荧光原位杂交,其杂交信号分布情况为：在较大的染色体(A亚组)几乎整条染色体上都有信号分布,尤其在染色体中部信号较强,为典型的较大染色体(A亚组)特异的重复序列；Pima90-53有丝分裂中期染色体以216B18作为探针,几乎在所有的染色体上都有信号分布,其中有6个较强信号分布在6条染色体的短臂端部,根据杂交信号来看,该BAC克隆主要是含有45S rDNA重复序列和一种分布于整个海岛棉染色体的散布重复序列。
3.只对其中的428K20和412A11分别构建了亚克隆文库。对亚克隆测序并进一步进行序列分析。序列比对结果显示：428K20含有陆地棉mGhCIR291、JESPR272、LIB5327-017-A1-N1-D12_P_S_Gh557类型微卫星相关序列、其大部分序列均为gypsy-类型反转座子(gypsy-like retrotransposon),而很可能正是这一类型gypsy反转座子,参加了由较大染色体(A亚组)向较小染色体(D亚组)的入侵过程；412A11则主要检测到了大量的预测基因、预测蛋白、表达mRNA及陆地棉-微型反向重复转座元件(miniature inverted repeat transposable element,MITE),根据A基因组染色体两端较强的杂交信号,推断A基因组染色体靠近端粒或亚端粒区域可能含有很多微型反向重复转座元件(miniature inverted repeat t ransposable element,MITE)。
In this study, the genome of G.barbadense was used to isolate specific repetitive sequence. Repeated sequence distribution in G.barbadense, G.arboreum, G.herbaceum, G.herbaceum var.africanum and G.raimondii were verified by FISH after constrction of subclones and sequence analysis.The main results were obtained as follows:
1.19,200 BAC clones were screened from pima90-53 BAC library. Fluorescence in situ hybridization (FISH) was carried out on G..barbadense mitotic metaphase chromosomes by using the probes of specific repetitive sequence.According to the distribution of hybridization signal, we found four BAC monoclonal.The clone numbers are:428K20,412A1l,334E15,216B18.
2. In situ hybridization results showed that 428K20 can produce strong signal in all the chromosomes of G.barbadense,428K20 produced a strong hybridization signal in besides the chromosome ends large chromosomes (A subgenome) of G.barbadense. However, hybridization signal could be found in the region near the centromere of small chromosomes (D subgenome).In G.arboreum, the signal distribution in accordance with large chromosomes (A subgenome)of G.barbadense but it can't produce hybridization signal in G.raimondii;422Al1 only was produced hybridization signal in large the chromosome(A subgenome) ends of G.barbaddense.It also could be produced hybridization signal in the chromosome ends of G.arboreum、G. herbaceum、Gherbaceum var.africanum. But it didn't produce hybridization signal in G.raimondii.It is therefore confirm that 428K20 and 412A11 are specific repetitive sequence of A genome,but 428K20 contains a transpositional element(sequence);334E15 produced hybridization signal in besides the chromosome ends large chromosomes(A subgenome)of G.barbadense,especially hybridization signal were very strong in the middle of large chromosome(A subgenome). Such BAC clones may be more relevant in the pima90-53 BAC library.It is a typical A subgenome specific repeat sequence;216B18 can produce hybridization signal in all the chromosome of G.Barbadense but six strong hybridization signal which are located at the ends of six chromosome. Such BAC clones may be also more relevant in the pima90-53 BAC library.According to hybridization signal,216B18 mainly contains 45S of repetitive sequence and a kind of interspersed repeat sequences which are distributed in all chromosomes of G.barbadense.
3.428K20 and 412A11 was only constructed subclone library. The result of subcloning sequencing data indicated that 428K20 were contain mGhCIR291、 JESPR272、LIB5327-017-A1-N1-D12_P_S_Gh557 of micro-satellite sequences and gypsy-like retrotransposon in G..hirsutum,Gypsy-like retrotransponson may attended invasion from large chromosomes(A subgenome)to small chromosomes(D subgenome). While 412A11 was primarily predicted genes predicted protein, mRNA expression and miniature inverted repeat transposable element (MITE) of G..hirsutum. According to hybridization signal of A genome chromosomes, it is inferred that telomere or subtelomeric regions of A genome chromosomes may be contain miniature inverted repeat t ransposable element (MITE).
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