Danian Huang

Map-based cloning of the ALK gene, which controls the gelatinization temperature of rice

Gelatinization temperature (GT) is an important parameter for evaluating the cooking and eating quality of rice besides amylose content (AC). The inheritance of the genes affecting GT has been widely studied and is considered to be controlled by a major gene. Here, we report the map-based cloning of rice ALK that encodes the soluble starch synthase II (SSSII). Comparison between the DNA sequences from different rice varieties, together with the results obtained with digestion of the rice seeds in alkali solution, indicates that the base substitutions in coding sequence of ALK may cause the alteration in GT.

QTL analysis of leaf photosynthetic rate and related physiological traits in rice (Oryza sativa L.)

Photosynthesis is the primary source ofdry matter production and grain yield inrice. Study on genetics of photosynthesisand related physiological characters isvery important to rice physiologicalbreeding. In this study, a double haploid(DH) population derived from anther cultureof ZYQ8/JX17, a typical indica and japonicahybrid was used as genetic material. Netphotosynthetic rate, chlorophyll content,stomatal resistance and transpiration rateof the parents and DH lines wereinvestigated in flourishing tilleringperiod. The quantitative trait loci (QTLs)for each trait were analyzed based on theconstructed molecular linkage map of thispopulation. A total of 8 QTLs forphotosynthesis and related physiologicalcharacters were detected. Two putative QTLsfor net photosynthetic rate (qNPR-4 andqNPR-6) were mapped on chromosome 4 and 6,respectively. Three QTLs (qCC-1, qCC-3 andqCC-8) for chlorophyll content weredetected on chromosome 1, 3 and 8,respectively. One QTL for stomatalresistance (qSR-4) was identified onchromosome 4. Two QTLs for transpirationrate (qTR-4 and qTR-7) were detected onchromosome 4 and 7, respectively. TheseQTLs should accelerate the process ofbreeding new rice varieties with higherphotosynthetic capacity and higher yield.

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R1 generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.

QTL analysis of rice low temperature germinability

A double haploid population, derived from anther culture of F1 hybrid between a typicalindica and ajaponica (ZYQ8/JX17), has been used to investigate the low temperature germinability (LTG) at 15°C. The low temperature germinability of two parents was significantly different. In 6–11 d, the germination percentage of ZYQ8 was higher than that of JX17. In 12–16 d, the germination percentage of JX17 was higher than that of ZYQ8. The quantitative trait loci (QTLs) of every day for low temperature germinability have been mapped based on a molecular linkage map constructed from this population. In 8–11 d, qLTG-9 was identified in C397B-RZ617B on chromosome 9, the additive effect was positive, showing that the allele from JX17 could increase low temperature germinability. In 12–16 d, qLTG-4 was mapped between RG908 and CT563 on chromosome 4, the additive effect was negative, showing that the allele from ZYQ8 could increase low temperature germinability. These two QTLs were detected at different stages, showing the complexity of the mechanism of low temperature germinability.

Hereditary Behavior of bar Gene Cassette is Complex in Rice Mediated by Particle Bombardment

Particle bombardment transformation using minimal gene cassette (containing the promoter, open reading frame and terminator) is the novel trend in plant genetic transformation, and its use helps to alleviate the undesirable effects of plasmid vector backbone sequences on transgenic plants. In the present article, studies related to the hereditary behavior of bar gene cassette in T(1) to T(3) generations of the transgenic rice (Oryza sativa L.) lines transformed by particle bombardment have been discussed. The selectable marker bar gene cassette that integrated with the rice genome had multiple copies and showed complex segregation behaviors including the presence of false homozygotes, with abnormal segregation ratios ranging from 35:1 to 144:1 (Basta-resistant: sensitive plants) in their progenies. In five out of ten original transgenic lines, bar gene can be stably transmitted as a dominant gene to self-pollinated T(2) progeny. The homozygotes were obtained in three transgenic lines in T(1) generation regardless of the multiple-copy integration patterns of bar gene. Southern blotting analysis showed that multiple copies of bar gene cassette were linked, which formed transgene arrays in the host rice genome. The authors also observed stable transmission of integration patterns of bar gene cassette, as obtained from Southern blotting analysis, in the regularly segregated transgenic rice lines and loss of gene in an irregularly segregated transgenic line. The segregation behavior varied among the transgenic progenies that exhibited similar Southern hybridization patterns of bar gene. On the basis of these results, the multiple-copy integration, gene lost, and gene expression interaction were the major reasons for the complex segregation behaviors of bar gene cassette in transgenic rice plants.

Salt tolerance of transgenic rice (Oryza sativa L.) with mtlD gene and gutD gene

Southern blot analysis indicated thatmtlD gene (encoding mannitol-1-phosphate dehydrogenase) andgutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated byAgrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.

New technology to examine and improve the purity of hybrid rice with herbicide resistant gene

The herbicide resistant bar gene has been widely used as selectable marker genes in the study on plant genetic transformation. Owing to the integration of the gene into rice genome, transgenic rice was resistant to the herbicide Basta. Therefore, selection of transgenic plant md genetic analysis became easier. In the studies, bar gene was introduced as a genetic marker gene into ria restore lie variety of two-line or three-line. Combined with conventional breeding method, the good herbicide resistant individual plant was derived as the new restorc line for hybrid combination. After sprayed with Basta at seeding phasc. the real hybrid rice that hnd herbicide resistance could grow normally, while the fnke hybrid rice and weeds, having no bar gene, were killed by herbicide. The method described above helped rapidly to examine and improve the purity of hybrid rice.

Relationship between CMS-specific mitochondrial structures and pollen abortive phenotype in rice CMS lines

Cytoplasmic male sterile (CMS) lines varied at pollen abortive phenotypes (PAP) have been used effectively for production of hybrid seeds in rice. To investigate the relationship between PAP of CMS lines and CMS-specific mitochondrial genes or structures in rice, CMS lines varied at PAP developed on 18 cytoplasmic sources were examined with seven PCR primers specific to three CMS-specific mitochondrial regions. The 18 CMS lines were classified into stained abortive (SA) and typical abortive (TA) types according to their PAP. Two CMS cytoplasms-specific PCR fragments, by which genotypes between the CMS lines and their maintainers were distinguished, were identified with primer LD29 or LD30. Furthermore, it was discovered that phenotypes of SA-CMS and TA-CMS were related to CMS-specific mitochondrial genes or structures, LD-orf310, atp6-orf79-like, and orf288-like structures. The results suggested that CMS phenotypes were mainly controlled by cytoplasmic genes, and it could be speculated that the SA-CMS phenotype was related to LD-orf310 and atp6-orf79-like structure, while TA-CMS phenotype was related to WA352.