葡萄果实发育后期基因表达水平的变化对果实品质形成具有重要影响,内参基因的选择是基因表达定量分析的关键影响因素。本试验以雷司令葡萄品种花后50、75和95 d的果实为材料,通过半定量RT-PCR,研究了常用持家基因Actin、Tubulin、GAPDH1、8 S rRNA的表达变化,探讨其作为葡萄果实发育后期基因表达半定量分析内参基因的可行性。结果表明:18 S rRNA的表达水平高,且最稳定,其次是Actin,而Tubulin和GADPH的相对表达水平较低;对VvTLP、GHF17 P和VvASR3个功能基因在不同果实材料中的表达水平进行半定量RT-PCR,VvTLP和VvASR的相对表达水平较高,GHF17 P的表达水平较低,整体上均呈现递增的趋势;以Actin和18 S rRNA为内参得到的归一化结果基本一致,在4个持家基因中18 S rRNA和Tubulin是优选的葡萄果实发育后期基因表达研究的内参基因。
Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.
Wuschel-related homeobox （WOX） genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOXgene family member from grape （Vitis vinifera L. cv. Cabernet Sauvignon）, and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem （SAM） of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L-1 2,4-D, 1 mg L-1 NAA and 1 mg L-1 BAP treatments, while downregulation was monitored by 1 mg L^-1 IBA treatment, and there was no response to 0.5 mg L-1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.
DAI RuJIN Hai-pengWANG ZengAvihai PerlXU Hai-yingZHANG WenCHEN Shang-wuMA Hui-qin
