Improvement Of Perennial Ryegrass (Lolium Perenne) Tolerance To Drought By Gene Transferring Of Stress-inducible Transcription Factors DREB1B And CBF1 Gene

Drought is the most important factor causing stress environment in the north-west area of china, and it seriously affects plant growth. The aims of this research are 1) to establish the breeding system of transgenic perennial ryegrass, and 2) to transfer the drought-tolerant genes into perennial ryegrass in order to enhance its tolerance ability of water stress.Four expression vectors of pBAC123 and pBAC128, pBAC122 and pBAC127 were constructed, and they all contain the drought-inducible expression promoter rd29B which could drive DREB1B gene or CBF1 gene ,respectively. Among them pBAC128 and pBAC127 contain the constitute expression promoter CaMV35S which could drive the selection marker {bar gene). The four expression vectors (pBAC122 and pBAC123 were co-transferred with p35SIH3 which has selection maker bar gene.) were transformed into embryogenic calli of perennial ryegrass (Lolium perenne) by biolistic bombardment system of PDS1000/He, respectively. Transformants were screened on selection medium with bialaphos. Finaly 98 transgenic plants were produced following plant regeneration culture.Genomic DNA's PCR and dot-blotting analysis were conduced to screen the transgenic plants. The results showed that foreign CBF1 gene and DREB1B gene were well integrated into some transgenic plant's genome of ryegrass, respectively. To test transgenic plants' resistance to herbicide PPT, five concentrations of bialaphos were used to daub leaves of transgenic plants. Some transgenic plants could resist bialaphos up to 135-200mg/L while wild type has no resistance.Under drought stress, the free proline contents of 31 lines among 63 DREB1B gene transgenic, and 13 lines among 36 CBF1 transgenic showed 2-4 times increasing compared with non-transgenic wild type. With 15% PEG 8000 to produce osmotic stress, free proline contents of transgenic lines increased 10~20 times while non-transgenic wild type showed only 5 times increase. Under drought stress for 25 days, 9 transgenic lines behaved green and living, and 4 transgenic lines restored living after watering.Our research suggested that we could greatly improve ryegrass tolerance to drought by transfer drought-inducible expression promoter rd29B which could regulate the expression of foreign DREB1B gene and CBF1 gene in ryegrass.