分子生物学文献

Experimental objective
1.Introduct an alien gene (SNAC1)into elite wheat cultivar to improve the tolerances of wheat cultivars to drought and salinity through genetic engineering. 2.Assays to confirm the tolerances of drought and salintity,sensitivities to ABA of transgenic wheat cultivars. 3. How SNAC1 regulate expression of genes in the transgenic wheat.
Materials and methods
1.Vector construction 2.Transformation and tissue culture 3.PCR, reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) 4.Southern blot analysis 5.Drought and salt tolerance assays 6.Abscisic acid (ABA) sensitivity assay 7.Experimental result
PCR, reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR)
DNA and RNA was extracted from young leaves of wheat. The selected transgenic plants were analyzed by PCR , primers S-SNA and NOSR were used to amplify a 392-bp DNA segment spanning SNAC1 gene and NOS-terminator. The PCR products were separated on 1.2% agarose gels.
PCR Amplification program:
Following a denaturation step at 95 ◦C for 4 min, the amplification was carried out with 40 cycles at 94 ◦C for 15 s, 60 ◦C for 20 s and 72 ◦C for 20 s.The fluorescent signals were recorded at 72 ◦C for 30 s. The experiments were performed in triplicates.
drought and salinity are the primary factors limiting wheat production worldwide. It has been shown that a rice stress-responsive transcription factor encoded by the rice NAC1 gene (SNAC1) plays an important role in drought and salt stress tolerance.
2.Increased sensitivities to abscisic acid
The ABA sensitivity of the transgenic wheat plants increase,the growth of roots and shoots are significantly inhibited in the transgenic plants
A rice stress-responsive NAC gene enhances tolerance of transgenic wheat to drought and salt stresses
The fuction of NAC1 gene
1.Improvement of drought and salinity stress tolerance in wheat.
Transformation and tissue culture
An elite Chinese wheat cultivar (cv.) Yangmai12 was planted in an experimental field,seeds at 14–16 days post anthesis (DPA)were isolated as explants. Embryos were isolated from the seeds,placed onto agar plate and cultured for 5 days, Calluses were used for bombardment with a biolistic gun, Subsequent cultures and selection of transgenic calli were performed as previously described,The surviving plantlets with strong roots and shoots were vernalized at 4 ◦C for 14 days and then cultured in a growth chamber under 16/8 h day/night at 18–20◦C.
Vector construction
SNAC1 was amplified from a rice cDNA clone using two primers SNAnde1 and SNAsac1 . The PCR product was cloned into pAHC25 (Ubiquitin promoter-based vectors)to generate a recombinant plasmid pUbiSNAC1 for wheat transformation .
Result
Transgenic calli were selected on medium containing phosphinothricin (PPT).A total of 18 PPT-resistant T0 transgenic plantlets were generated and used for the production of T1 progenies. For T1 to T3 generations, PPT was used for the selection of transgenic plants. Two transgenic lines showed good performance under drought and salt treatment ,They were designated as A2 and A4 and used for subsequent analyses.A 392-bp SNAC1 fragment was amplified from A2 and A4 using primers S-SNA and NOSR.RT-PCR analyses confirmed the presence of SNAC1 transcripts in the two transgenic lines with the same primers , while internal control actin transcripts were present in all transgenic plants and the non-transgenic Y12 plants. Southern blot analyses revealed that the SNAC1 gene was indeed integrated into the wheat genome with different numbers of hybridization bands in the two lines, suggesting that A2 has at least one copy and A4 has at least 2 copies.
Southern blot analysis
A total of 15 μg DNA from each sample was digested with 60 units of SacI overnight, and the digested DNA was electrophoresed on 0.8% agarose gels. DNA fragments were transferred onto a nylon membrane (Hybond-N+, Amersham,UK), and then hybridized with a-[32P]-dCTPlabeled DNA fragments of ubiquitin promoter amplified with primers UbiP5 and UbiP6. Autoradiograph was recorded and analyzed with an imaging analysis system .
qRFra Baidu bibliotek-PCR
qRT-PCR system:
reaction containing SYBR Green I PCR Master Mix.(25μL) diluted cDNA (1:100). (10μL) forward and reverse gene-specific primers:Gene-specific primerswere designed using Primer Premier5 software.(10 pmol each of primer) primers ActinF and ActinR:Wheat actin gene was selected as an internal control and co-amplified to normalize the total amounts of cDNA present in each reaction. PCR amplification was performed in Lightcycler® 480
RT-PCR
For RT-PCR analyses of a target gene, 5 μg total RNA extracted from wheat leaves were reversely transcribed into cDNA with reverse transcriptase and an oligodT20 primer. PCR amplifications were performed with a thermal cycler using primers SSNA and NOSR. PCR consisted of 35 cycles of denaturation (94 ◦C,30 s), annealing (60 ◦C, 30 s) and extension (72 ◦C, 30 s). Wheat actin gene was co-amplified as control with primers PwactF1 and PwactR1