Gene Cloning Of ADP-ribosylation Factor From Gossypium Hirsutum L.

Cloning the specific genes expressed during anther development is immensely important, not only for elucidating the molecular biological mechanism of plant male sterility, but also for accelerating its application in heterosis through genetic engineering method.Dong A, the male sterile line of cotton discovered in 1972 from a pollenless mutant of cotton variety Dongting No.l, is an ideal materials used to study the pollen-specific genes during anther development. The fertile individuals and its pollenless mutant have a highly homogeneous genetic background except for the male sterility. Using cDNA-AFLP technique, gene expression of the fertile of Dong A and its male sterile individuals in anther development stage were compared. A fragment, GHA27, was found to be similar to ADP-ribosylation factor (ARF) of Arabidopsis thaliana, Triticum aestivum, etc. In this paper, the complete cDNA of GhAJZF was isolated from a cotton cDNA library using GHA27 as probe. The maui results were as follows:1. Gene cloningDifferential fragment GHA27 was produced by cDNA-AFLP method employed to compare the gene expression in developing anthers between the male sterile and fertile plants of cotton Dong A. A cDNA clone, designated GhARF (Gossypium hirsutum ADP-ribosy lation factor), was isolated from a cotton (Gossypium hirsutum L.) cDNA library using GHA27 as probe.2. Sequence analysisThe cDNA clone contains an 546bp ORF (open reading fragment) encoding a predictedprotein of 181 amino acids with molecular weight of 20.7kD, 130bp 5'non-coding region and 152bp 3' non-coding region including polyadenylation signal sequence AATTAA and poly A tail.3. Homology analysisBLAST analysis showed that the amino acid sequence of the ASF gene cloned fromcotton was significantly homologous with ADP-ribosylation factor (ASF) of mammalian,plant and yeast, etc. The amino acid sequence of the gene shows 99% (179/180) , 98%(177/180) ,96% (174/180) and 92% (168/180) identity to ArabidopsisthalianaARFl,Triticum aestivum, Solatium tuberosum and Bos Taurus, respectively.4. Conserved domain of amino acidThe deduced amino acid sequence showed that the 20.7kD protein encoded by the ARF gene had consensus GTP-binding domain P(GLDAAGKT), G'(DVGGQ) and G(NKQDL).5. Expression analysisThe expression analysis of GhARF in different tissues of cotton was carried out by RNA dot blotting with GhAKF probe labeled with DIG. The result showed that the expression of ARF gene was mainly in anthers of sterile and fertile, pollen of fertile, corolla rather than in leaves, radicel and ovule.6. Southern blottingCotton genomic DNA was digested with Bglll, Kpn I , Hindlll, EcoRVand EcoR I respectively and hybridization was carried out with GhARF probe labeled with DIG. Southern blotting analysis indicated that there were at least two copies encoding ARF gene in the genome of cotton.