Cloning Of Cotton Black Spot-Related Genes And Investigation On The Disease Early Diagnosis

Fungus Alternaria macrospora is the causal pathogen of Cotton Black Spot(CBS) disease on cotton seedlings from cotyledon period to seedling stages causing black spot on leaves and their damping-off.Endopolygalacturonases(endoPGs)place a major role on pathogen penetration on plants at the initial phase of invasion digesting the pectic polysaccharides on the plant primary cell walls used by plants as potential barrier to fight against pathogens.Plant polygalacturonase-inhibiting proteins(PGIPs)play an important role on defense system for effectively and specifically binding to and inhibiting endoPGs secreted by pathogen during invasion.Therefore,study on the genes encoding the two macro-moleculars is considered as a key way to disclose the mechanism of plants defensing fungal infection.In addition,an initiation was launched to try to establish early diagnosis on CBS by using pgip gene of cotton as probe with chlorophyll fuorescence technique as assistant.In this regard,researches have been implemented with experimental results stated as below:The gene encoding endoPGs designated ampg from A.macrospora was cloned and characterized by using a novel method on primers design with open reading frame(ORF) 1242bp in size.Sequence alignment showed that it shared quite high identities with two another Alternaria fungi:Alternaria alternata and Alternaria cirri(98.4%and 98.3%, respectively.)while its deduced peptide sequence was much low identical to the two fungi(both 76%).Comparing the nucleotide sequences from genome and RNA revealed that there was no intron in ampg.Analysis of nucleotide sequence of ampg displayed it has two more fragments about 50bp each compared with that of the other two Alternaria causing an important protein motif(H-x-x-S-x-x-S)for enzyme activity in its predicted protein(AMPG)more flexible thus probably less effective.That could be a part of bases to evaluate the evolution of fungi Alternaria according to the basic evolution principle that species evolve towards the direction promoting their survival and good life conditions.Secondary structure prediction plots showed that AMPG belongs to Glycosyl hydrolases family 28 and has two PbH1 domains,the characteristic motif for endoPGs,one potential N- glycosylation site and five potential O-glycosylation sites.A gene encoding PGIPs in cotton(Gossypium hirsutum)named Ghpgipl was cloned and sequenced showing that it had 993bp in length and its predicted translational product was 330 amino acids in size with a molecular mass of 37.0 KDa and pI of 8.30. Compared with that of other 9 species involved the sequence homologies were from 65.6%to 70.3%on gene level and from 63.4%to 68.6%on amino acid level.The deduced protein sequence analysis revealed that there were 6 potential N-glycosylation sites and 10 cysteine residues N- and C- in total.Sequence alignment of deduced amino acid of Ghpgipl with that of the other 9 species indicated that there were 5 conservative leucine rich repeats(LRR)domains,characteristic of proteins with defense ability and most of Leucine in them was highly unchanged.Semi-quantitative RT-PCR disclosed that Ghpgipl were constitutively expressed whether in root,stem or leaf and the amount in leaf is most heavy than the other two.There is no report on early diagnosis of CBS,one of three diseases that are most devastating to cotton in its seedling phase.Based on expression of the defensing gene in cotton that encodes polygalacturonase-inhibiting proteins,Semi-quantitative Reverse Transcription Polymerase Chain Reaction(Sq RT-PCR)technique was applied on early diagnosis of this disease.For more precise and cogency,Information in photo from Sq RT-PCR technique was exchanged into that in data by professional imaging process software Sigmascan Pro.Sq RT-PCR indicated that the gene had little change in the amount of its expression before the fifth day after invasion with Alternaria macrospora and its number was rapidly decreasing after the time.That was proved by the result using chlorophyll fluorescence technique that the PSâ…¡operating efficiency and electron transport rate of photosynthesis also had small changes before that time and then was dramatically decreasing.Therefore,when Alternaria macrospora infects cotton,there would be a short-time(five days)defense system functioning and they would successfully invade after the defensing time over.That could be important guidance on poisoning and prevention of CBS.