Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.
Jiahua Xie, Todd C. Wehner, and Mark A. Conkling
I.K. Kang, D.A. Starrett, S.G. Suh, J.K. Byun, and K.C. Gross
We are studying β-galactosidase (EC 184.108.40.206) in softening persimmon fruit (Diospyros kaki L.f. cv Fuyu) and hope to decrease the rate of softening by inserting an antisense construct of the β-galactosidase gene. The N-terminal amino acid sequence of persimmon fruit β-galactosidase was recently reported. Here we report the cloning of a putative β-galactosidase gene from persimmons. Degenerate oligonucleotide primers were synthesized based on the amino acid sequence. 5′-RACE (rapid amplification of cDNA ends) was done using persimmon Poly A+ mRNA extracted using a phenol: chloroform/LiCl method. Purification was done on an oligo dT-cellulose column. A fragment of roughly 150 base pairs was purified by agarose gel electrophoresis and subcloned into the pCR-Script cloning vector from Stratagene. After sequencing and verifying the insert's identity, it will be isolated and used to screen a persimmon fruit cDNA library currently being constructed. Ultimately this cDNA clone will be used to make an antisense β-galactosidase construct that will be transformed into persimmon.
H.P.V. Rupasinghe, K.C. Almquist, G. Paliyath, and D.P. Murr
We tested the hypothesis that conversion of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) to mevalonate (MVA) catalyzed by HMG CoA reductase (HMGR) is the rate limiting step for α-farnesene biosynthesis of apples. In higher plants, isopentenyl pyrophosphate (IPP) is derived via two pathways: 1) the classical mevalonate pathway, and 2) the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR action. When apple skin discs were incubated with MVA, or GAP and pyruvate, MVA increased α-farnesene levels in the skin but not GAP and pyruvate. Treating apple fruits with Lovastatin (1000 ppm), a competitive inhibitor of HMGR, inhibited α-farnesene accumulation in the skin by 20% to 50% during storage. Content of α-farnesene in the skin increased during the first 2 to 4 months in storage, and then decreased. In contrast, HMGR activity, as determined by the conversion of [4-3H]HMG CoA to MVA in the total membrane and soluble fraction, was the highest at the time of harvest and gradually decreased during 5 months of storage in air at 0 °C. The potent ethylene action inhibitor 1-MCP inhibited ethylene production and α-farnesene evolution by 99% and 97%, respectively. The effect of 1-MCP on in vitro activity of HMGR was marginal (≈30% inhibition). 1-MCP inhibited respiratory CO2 evolution by 50%, which suggests also that inhibition by 1-MCP of α-farnesene synthesis in apple could be regulated by the acetyl CoA pool. In plants, HMGR is encoded by a small gene family and differentially expressed. As the first step of studying the molecular mechanism of HMGR regulation, we have isolated a 444-bp fragment of apple hmgr gene using apple skin mRNA and degenerate oligonucleotides designed against conserved regions of plant hmgr genes.
Kelly J. Vining, Q Zhang, C.A. Smith, and T.M. Davis
Shen, K.A. Meyers, B.C. Nurul Islam-Faridi, M. Chin, D.B. Stelly, D.M. Michelmore, R.W. 1998 Resistance gene candidates identified by PCR with degenerate oligonucleotide primers map to clusters of
Nobutaka Shiraiwa, Kaori Kikuchi, Ichiro Honda, Masayoshi Shigyo, Hiroko Yamazaki, Daisuke Tanaka, Kenji Tanabe, and Akihiro Itai
reaction (PCR) using two degenerate primers; sense: 5′-ATGTGGYMNGARGGNTTYAC-3′ and antisense: 5′-GTRTGNGCNGCNAGNCCCAT-3′ (Y = C/T, M = A/C, N = A/C/G/T, R = A/G). Degenerate oligonucleotide primers were designed from conserved regions of A. thaliana GA4
Shaolan Yang, Changjie Xu, Bo Zhang, Xian Li, and Kunsong Chen
our previously published protocol ( Xu et al., 2004 ). Degenerate sense primer EXPSP, 5′-ACAATGGGNGGDGCDTGTGG-3′ (D = A/G/T, N = A/C/G/T) and antisense primer EXPAP, 5′-TGCCARTTYTGNCCCCARTT-3′ (R = A/G, Y = T/C) were designed according to the conserved
Zhengke Zhang, Runshan Fu, Donald J. Huber, Jingping Rao, Xiaoxiao Chang, Meijiao Hu, Yu Zhang, and Nina Jiang
degenerate sense primer (5′-GSNCAYGCNACNTTYAYGGNG-3′) and an antisense primer, (5′-YTGCCARTTYTGNCCCCARTT-3′) (N = A/C/G/T, Y = C/T, N = G/C, R = A/G), which were designed according to the conserved regions of plant expansin sequences. The PCR product was
Akihiro Itai, Takaaki Igori, Naoko Fujita, Mayumi Egusa, Motoichiro Kodama, and Hideki Murayama
of total RNA from O-276-inoculated ‘Nijisseiki’ leaves using M-MLV reverse transcriptase (ReverTra Ace; Toyobo, Tokyo, Japan). The ACC synthase gene was amplified from cDNA by polymerase chain reaction (PCR) using the following degenerate
Matthew A. Escobar, Andrew Shilling, Pine Higgins, Sandra L. Uratsu, and Abhaya M. Dandekar
., 1983 ). To isolate a fragment of a PPO-encoding gene from the DNA, degenerate oligonucleotides were designed based on the highly conserved CuA and CuB copper-binding domains in plant PPOs. The primers Walt 2 (5′-AAR GAR GCI GAY MGI CCI GCI GGI GCI YTI