Isolation And Expression Analysis Of Gene Encoding PM-Type Ca～(2+)-ATPase

In agriculture, the pathogens cause severe losses in yield throughout the world. And continuousapplications of chemicals lead to heavy contaminations of the products and the environment. Duringthe past 20 years,β-Aminobutyric Acid (BABA) has been proved to be effective to induceresistance against a broad spectrum of pathogens in agricultural crops and vegetables. Moreover,BABA has been verified one of the most effective elicitors to introduce tolerance to abiotic stresses.Although the phenomenon has long been known, the underlying mechanisms are poorly understoodat the biochemical and molecular level. New methods should be employed to analyze the genefuncations, in order to better understand the induced resistance by BABA and the applicationpotential of BABA induced resistance.cDNA amplified fragment length polymorphism analysis (cDNA-AFLP) was used to revealtranscripts whose expression is rapidly altered during the treatment of soil drench ofβ-Aminobutyric Acid (BABA) to tomato (Lycopersicon esculentum L) roots.The main results are following1. 3 kits were used for RNA extraction from 3 tomato tissues, including leaf, stem and root,were evaluated upon quality,yields of RNA isolated. RNAplant was found to be the most effectiveone among the 3 reagents evaluated. With this kit, higher amount and quality of RNA could besuccessfully extracted from all 3 kinds of tissues tested.2. After treated with 25mmol/L BABA, cDNA-AFLP was used to reveal transcripts whoseexpression is rapidly altered during the treatment of soil drench of BABA to tomato roots, withDDH₂O as control. Among 5,000 transcript-derived fragments (TDFs), 23 showed increasedabundance and 6 showed decreased abundance. By cloning and sequencing, the sequences of 12TDFs were obtained. A high percentage of the differentially regulated TDFs identified in this studyhas homology to genes commonly regulated during plant defense/stress responses. According to thefunctions of homology genes, these 12 TDFs could be divided into 5 groups.â‘ BIF13 and BIF 14: members of P69, a pathogenesis-related proteins (PR);â‘¡BIF 2, BIF 3,6,17 and BIF 4: high homologies to genes all responsible for transductionacross membrane system of little molecular materials such as H₂O, Ca²⁺ and anti-fungal agents.â‘¢BIF5: signal transduction proteins, high homology to Ca²⁺-binding protein which is animportant dement in Ca²⁺ signaling pathway.â‘£BIF1: molecular chaperone protein, keeping activity of certain proteins under stresscondition.⑤BIF10, BIF and BIF 12: no homology to known genes.3. With the RACE and PCR technology, a PM-type (Plasma membrane-type) Ca²⁺-ATPasegene was isolated. The full-length cDNA is 3389 bp, and the open reading frame (ORF) is 3090bp,encoding 1029 amino acid. Both RT-PCR and the analysis of sequence, including Blastn,Blastx and ORF confirmed it is the first time that PM-type Ca²⁺-ATPase was isolated in Solanaceae plant.4. Northern Blotting was performed to reveal transcript level of PM-type Ca²⁺-ATPase indifferent tissues at different times. The result indicated that Ca²⁺-ATPase was abundant in root, lessin leaf and stem; furthermore, the transcript level of target gene was up-regulated and reached itspeak at 6 hours after the treatment, and it nearly could not transcript after 72 hours.5. The deduced amino acid sequence showed high identities with PM-type Ca²⁺-ATPase, buthad low identities with ER-type Ca²⁺-ATPase. Amino acid analysis with TMpred software indicatedthat the polypeptide chain contained 7 motifs,4 domains and 8 transmembrane helixes, which wererelated to the activity of the putative protein. The gene take participate in calcium signaling pathway.6. A full-length cDNA library induced by BABA was constructed by using SMART(switchingmechanism at 5' end of RNA tanscript) and Primer Extension techniques. The titers of unamplifiedand amplified cDNA library were 4.4×10⁶ pfu/mL and 1.8×10¹⁰ pfu/mL respectively. The rate ofrecombination was 96%. The average size of inserted fragments was 700-1000bp.7. A pair of degenerate primers was designed against the conserved regions that was used forSemi-quantitive RT-PCR to amplify cDNA segment from two citrus genotypes (Citrus unshiu Marcand Poncirus trifoliate) treated with low temperature. The results revealed that Ca²⁺-ATPase wasup-regulated by low temperature in citrus, suggesting this gene participating in adversity signalingconduction.