Enhanced Resistance To Blast Fungus Magnaporthe Grisea By Expression Of A Resistance Gene Pi-d2 In Transgenic Rice And The Study Of A Striped Mutant With Abnormal Floral Organs In Rice

â… . Enhanced resistance to blast fungus Magnaporthe grisea by expressionof a resistance gene Pi-d2 in transgenic riceRice blast, which is caused by the fungal pathogen Magnaporthe grisea, is one of the mostdevastating diseases of rice, so it is called cancer of rice. The measures of control to the fungi diseaseare mainly to use the bactericide and the resistant varieties. Practice proved that the most economicefficient and essential way to fight against blast is breeding and use of resistant varieties. The rise ofplant genetic engineering offers the wide selection to control the fungi disease. The technologyintroduces the foreign resistant genes into cultivar and develops the gene source, which provides a newway to crop resistant breeding.In rice, six R genes including Pi-b,Pi-ta,Pi-9,Pi-2,Piz-t and Pi-d2 had been cloned. The former5 resistant gene proteins belong to the NBS-LRR type proteins, while the Pi-d2 protein with a specialstructure B-lectin is regarded a new type protein. In the research, we firstly introduce the Pi-d2 geneinto susceptible varieties LTH and Nipponbare and resistant variety Zhonghua 9 withagrobacterium-mediated transformation. After the molecular test, GUS histochemical assay andresistance test, some plants with high resistance to rice blast have been obtained, which establish thefoundation of molecular breeding for rice and other gramineous plants and provide gene resources forhybrid breeding. The main results were as follows:1 Through agrobactetium-mediated transformation with the callus from immature embryos and matureembryos of different rice varieties, three difference expression vectors including pCB6.3kb whichharbored the 3.2kb-length native promoter and genome sequence of Pi-d2, pCB6.3kb that contained the2.2kb-length native promoter and genome sequence of Pi-d2, in pZH01-2.72kb full length of cDNAsequence of was driven by promoter 35S and the negative control vector pZH01-Tp309 were introducedinto rice. Some hygromycin resistant plants containing Pi-d2 were obtained.2 PSK-a, a biologically active peptide acting as a growth factor, was added into co-culture medium.The results showed that PSK-a indeed affected the recovery of resistant calli and the transformationfrequency of rice varieties. PSK-a with the concentration of 10 nmol/L could increase resistant calliand efficiency of transformation, with a 11% top increase, than the control. However, PSK-a with theconcentration of 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effectof PSK-a on agrobacterium-mediated transformation is related with the concentration of 2, 4-D inselection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSKplus 2 mg/L 2, 4-D than those treated with 1 mg/L 2, 4-D.3 PCR, RT-PCR, Southern blotting testing indicated that the foreign target gene randomly inserted into rice genome with 1-4 copy and stably inherited. The hygromycin resistance analysis of transgenic plantsprogeny showed that the hygromycin marker in many transformats was inserted into rice genomes withsingle loci and one copy, and already inherited to T₂ generation, and some pure hygromycin-resistancelines were obtained.4 The results of the inoculation with the rice blast race ZB15 in field and indoors showed that thetransgenic plants from T₁ enhanced the resistance to rice strain ZB15, the size and number of the lesionon leaf decreased, some populations showed the resistant and susceptible separation with Mendeliansegregation or non- Mendelian segregation. The resistant expression of foreign gene from threedifference expression vectors to ZB15 showed no significant difference. The transgenic lines from T₂progeny also showed susceptible-resistant segregation to ZB15. The results are corresponding withthat of T₁ progeny.5 Continuous field evaluation in three years showed that some transgenic plants enhanced the resistanceto neck blast, and obtained some high resistance lines to rice leaf blast and neck blast.6 The receptor cultivars and 9 transgenic stable lines derived from at less T₅ generation transgenic plantsharboring Pi-d2 gene were challenged in greenhouse by inoculating 39 isolates of Magnaporthe griseafrom Sichuan Province. The different transgenic rice lines had different resistant spectrum. The Pi-d2gene expressed in susceptible receptors evidently enhanced the disease-resistance frequency at above90%, while the disease-resistance frequency of transgenic plants from ZH9 almost didn't increase. Fourtransgenic homozygous lines harboring full length cDNA sequence of Pi-d2 gene were inoculated with58 isolates selected by Agricultural Science Research Institute of China. The results showed thattransgenic lines resisted more than 81.48% isolates.7 Analysis of resistant expression of transgenic plants haboring the intetest gene from three differentvectors showed that the native promoters about 2.2kb or 3.2kb of Pi-d2 had the fuction of promoter as35S. The resistance between transgenic plants with genome sequence of Pi-d2 and transgenic plantswith Pi-d2 full length cDNA had no difference.8 The results of investigation of the agronomic traits of transgenic plants showed that variation occurredin a few transformants and many plants didn't obviously change. Nutrition test of a transgenic plantsselected randomly indicated that the nutrition in transgenic rice didn't significantly change.â…¡. The study of a striped mutant with abnormal floral organs in riceRice is the model of monocotyledon. The study of rice development has important values in theoryand applications. In the study, rice double mutant without the alien gene derived from the transgenicprocess was found and researched in morphology, histological characters and genetic analysis, so foundation is constructed for the cloning of the mutant gene. The results were as follows:1 The phenotype characters of mutant: The mutant showed white stripe on stem, leaf and spikelet. Insome growing stage, the leaf started to produce fork or curliness. The floret number increased,showing the multi-lemma/palea, palea-like and lemma-like lodicules or enlarged lodicules,additional pistil and stamen and the spited floret. The extreme types showed that the a few newflowers or spikelets derived from floret.2 The physiological characters: The contents of chlorophyll and net photosynthesis rate in the mutantwere obviously lower than in the wild type. At the three growing stages including tilling stages,before heading and after heading stages, the activity of SOD, POD and CAT related the expressionof mutant characters of mutant.3 Cytological identity: With observation of cell ultra structure using electron microscope, the whitetissue showed concaved cell wall and abnormal plastid which can not develop normal lamellae andthylakoid. The transition tissues aside the green tissues had the bigger cells with almost normalchloroplasts, while the tissues aside the white tissues showed that the cells were small and thechloroplast grana had inordinate arrangement. The cells in green sectors grow normally. Themorphogenesis of floral organs was observed by using the scanning electron microscopy (SEM).Results showed that palea and lemma normal developed, then the development programmes weredifferent between wild type and mutant. Additional palea and lemma begun to format and theflower primordium splited or formatted trifid primordium. The large part of primordium firstdeveloped. The lodicuale primordium appeared between stament primordium and palea/lemmaprimordium, and developed into palea/lemma-like lodicules. Stamen development was notsynchronal and the sizes of stamen primordium were different in mutant. The size of the floretmeristem was bigger than that of wild type, and some floret meristem had permanent activation.4 Histological characters: the mesophyll cells of white tissue were dyed light, and the contortedleaves had the excess growing vascular bundle cells or increscent bulliform cells.5 Genetic analysis: the results of the analysis with the progeny derived from the mutant crossing withnormal varieties showed that the mutant characters controlled by cytoplasts gene and inhered withmaternal inheritance way. Chloroplast genome DNA was extracted, and was amplified with 180RAPD primers. Two difference fragments between mutant and wild type were obtained.