| Sulphur-containing amino acids (SAAs) play an imimportant role in the efficiency of wool growth in sheep and milk production in dairy animals. The forage rich in sulphur-containing amino acids is benefit to the wool growth, the increase of the content of sulphur in wool, and the enhancement of yield of milk. The forage plants are deficient in sulphur-containing amino acids became the first limiting amino acids, especially cysteine and methionine. Breeding of forage variety containing sulphur-rich amino acid is one of research focus on improvement of forage quality at present.Crowtoe (Lotus corniculatus L.) is one of the most main legume forages which contain little sulphur-containing amino acids. The sulphur-rich protein encoding genes are transferred into crowtoe to improve the content of the sulphur-containing amino acids of forage protein through genetic engineering. Zeins, maize prolamin, the major protein reserves of maize kernels, are accumulated in protein bodies (PBs) arising from the rough endoplasmic reticulum. Zeins can be classified intoα-,β-,γ- andδ-zeins.β-,γ- andδ-zein are rich in the essential sulphur amino acids methionine and cysteine. The zeins are characterized by their stabilization in ruminal and digestibility in intestines, so it can be utilized in forage quality improvement. Though this may be considered a good starting point, further improvements must still be made to significantly increase the nutritional quality of forages, considering that foreign proteins must account for 1%～5% of the total protein in the leaf .The sulphur-containing amino acids genes ofγ-zein and zeolin (fused withγ-zein and phaseolin ) were fused with the report gene GFP from the plant expression vector pCAMBIA1302. The plant expression vectors pCBzeolin and pCBzein were obtained. pCBzeolin and pCBzein were transformed into the model plant tobacco by leaf disc method to prove the validity of pCBzeolin and pCBzein. The expressions ofγ-zein and zeolin gene from transgenic tobaccos were analysesd. pCBzeolin and pCBzein were also transformed into Lotus corniculatus mediated by strain LBA4404. The transformed regeneration plants were testified by molecular detections and analysis of the content of sulphur-containing amino acid. The main results of this study are as following.1. Zeolin gene (about 1600bp) andγ-zein gene (about 720bp) were got by cutting from the middle vectors pMD18zeolin and pMD18zein respectively, and were linked to the expression vector pCAMBIA1302 in the same endonucleases digestion sites. The plant expression vector pCBzeolin and pCBzein were obtained,sequenced and analyzed by bioinformatics software.2. Plasmids pCBzeolin and pCBzein were transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and then were transformed into tobacco and crowtoe mediated by strain LBA4404.3. After selective culture and PCR analysis, transgenic tobacco regeneration plants were obtained. It was showed thatγ-zein and zeolin accumulated in protein bodies were distributed heterogeneously in the protoplast to prove the validity and feasibilityof pCBzeolin and pCBzein.4. After co-culture, selective differentiation and regeneration, resistant crowtoe plants were obtained. Resistant plants were detected by PCR, RT-PCR and Dot blot. The results have proved that theγ-zein and zeolin genes have been transformed into the genome of L. corniculatus, and expressed on nucleic acid level in the transgenic plants.5. Sulphur-containing amino acid of transgenic crowtoe plants analysis showed that the content of sulphur-amino acid of transgenic zeolin crowtoe plants was significantly higher than that of the content of the transgenicγ-zein crowtoe plants and non-transgenic crowtoe plants (P<0.01), but there was no significant difference between the transgenicγ-zein plants and non-transgenic plants.
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