Grula J W, Hudspeth R L, Hobbs S L, Anderson D M
Plant Mol Biol. 1995 Aug;28(5):837-46. doi: 10.1007/BF00042069.
The acetohydroxyacid synthase (AHAS) gene family of the cotton AD allotetraploid Gossypium hirsutum has been cloned and characterized. We have identified six different AHAS genes from an analysis of genomic clones and Southern blots of genomic DNA. Four of the six genes are organized as tandem pairs, in which the genes are separated by only 2-3 kb. Conservation of restriction fragment length polymorphisms between G. hirsutum and A-genome and D-genome-containing diploid cottons was sufficient to assign the single genes in clones A5 and A19 to the A and D subgenomes, respectively. Each diploid genome has one tandem pair, but in these cases we could not make specific subgenomic assignments. DNA and deduced amino acid sequences were determined for the A5 and A19 genes, and an AHAS cDNA clone isolated from a leaf library. The sequence of the A19 gene matches that of the cDNA clone, while the A5 gene is 97.8% similar. The four genes comprising the tandem pairs are much less similar to the cDNA clone. The deduced amino acid sequences of the mature polypeptides encoded by the A5 and A19 genes are collinear with the housekeeping forms of AHAS from Arabidopsis thaliana, Nicotiana tabacum and Brassica napus. The constitutive expression of A5 and A19 was confirmed with RNase protection assays and northern blots. We conclude that these genes encode the main housekeeping forms of AHAS in G. hirsutum. Among the four AHAS genes comprising the two tandem pairs, at least two are functional. These genes exhibit either low-level constitutive expression (one or both of the 'downstream' genes of each pair), or highly specific expression in reproductive tissue (one or both of the 'upstream' genes of each pair). The AHAS gene family of G. hirsutum is more complex than that of other plants so far examined.
已克隆并鉴定了棉花异源四倍体陆地棉的乙酰羟酸合酶（AHAS）基因家族。通过对基因组克隆和基因组DNA的Southern杂交分析，我们鉴定出了6个不同的AHAS基因。这6个基因中的4个以串联对的形式排列，其中基因之间仅相隔2 - 3 kb。陆地棉与含A基因组和D基因组的二倍体棉花之间限制性片段长度多态性的保守性足以将克隆A5和A19中的单个基因分别归属于A和D亚基因组。每个二倍体基因组都有一个串联对，但在这些情况下我们无法进行具体的亚基因组归属。测定了A5和A19基因的DNA和推导的氨基酸序列，并从叶文库中分离出一个AHAS cDNA克隆。A19基因的序列与cDNA克隆的序列匹配，而A5基因与之相似度为97.8%。构成串联对的4个基因与cDNA克隆的相似度要低得多。A5和A19基因编码的成熟多肽的推导氨基酸序列与拟南芥、烟草和甘蓝型油菜的看家型AHAS是共线的。通过核糖核酸酶保护分析和Northern杂交证实了A5和A19的组成型表达。我们得出结论，这些基因编码陆地棉中AHAS的主要看家型。在构成两个串联对的4个AHAS基因中，至少有两个是有功能的。这些基因要么表现出低水平的组成型表达（每对中“下游”基因中的一个或两个），要么在生殖组织中表现出高度特异性表达（每对中“上游”基因中的一个或两个）。陆地棉的AHAS基因家族比迄今为止所研究的其他植物的更为复杂。
