利用FITC(fluorescein is othiocyanate,异硫氰酸荧光素)标记的外源DNA对小麦进行了花粉管通道法转化,在荧光显微镜下观察了外源DNA进入小麦胚囊的情况,并初步研究了转化时间及转化溶液组成对DNA进入胚囊效率的影响。结果表明,外源DNA沿着花粉管生长过程中形成的花粉管通道进入胚囊;授粉45 min和60min进行转化外源DNA进入胚囊的几率较大,因此在此段时间开始转化较为合适;相对于TE缓冲液,将0.05%Silwet L-77和5%蔗糖作为转化溶液可以缩短DNA进入胚囊的时间,但不能提高外源DNA进入胚囊的几率。研究表明,使用FITC标记观察外源DNA进入胚囊效率的方法,可简便有效地应用于花粉管通道法转化条件的初步优化。
Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.
