Zhu Yingguo

Discovery of mitochondrial chimeric-gene associated with cytoplasmic male sterility of HL-rice

The mitochondrial genome libraries of HL-type sterile line (A) and maintainer line(B) have been constructed. Mitochondrial gene, atp6, was used to screen libraries, due to the different Southern and Northern blot results between sterile and maintainer line. Sequencing analysis of positive clones proved that there were two copies of atp6 gene in sterile line and only one in maintainer line. One copy of atpt6 in sterile line was same to that in maintainer line; the other showed different flanking sequence from the 49th nucleotide downstream of the termination codon of atp6 gene. A new chimeric gene, orfH79, was found in the region. OrfH79 had homology to mitochondrial gene cox II and orf107, and was special to HL-sterile cytoplasm.

The breeding of two polyploid rice lines with the characteristic of polyploid meiosis stability.

Polyploidization is a basic feature of plant evolution. Nearly all of the main food, cotton and oil crops are polyploid. When ploidy levels increase, yields double; this phenomenon suggested a new strategy of rice breeding that utilizes wide crosses and polyploidization dual advantages to breed super rice. Because low seed set rates in polyploid rice usually makes it difficult to breed, the selection of Ph-liked gene lines was emphasized. After progenies of indica-japonica were identified and selected, two polyploid lines, PMeS-1 and PMeS-2 with Polyploid Meiosis Stability (PMeS) genes were bred. The procedure included seven steps: selecting parents, crossing or multiple crossing, back-crossing, doubling chromosomes, identifying the polyploid, and choosing plants with high seed set rates that can breed themselves into stable lines. The characteristics of PMeS were determined by observing meiotic behaviors and by cross-identification of seed sets. PMeS-1 and PMeS-2, (japonica rice), have several characteristics different from other polyploid rice lines, including a higher rate of seed set (more than 65%, increasing to more than 70% in their F1 offspring); and stable meiotic behaviors (pairing with bivalents and quarivalents nearly without over-quarivalent in prophase, nearly without lagging chromosomes in metaphase and without micronuclei in anaphase and telophase). The latter was obviously different from control polyploid line Dure-4X, which displayed abnormal meiotic behaviors including a higher rate of multivalents, univalents and trivalents in prophase, lagging chromosomes in metaphase and micronuclei in anaphase and telophase. There were also three differences of the breeding method between PMeS lines and normal diploid lines: chromosomes doubling, polyploidism identifying and higher seed set testing. The selection of PMeS lines is the first step in polyploid rice breeding; their use will advance the progress of polyploid rice breeding, which will in turn offer a new way to breed super rice.

Expression, Purification, and Secondary Structure Prediction of Pentatricopeptide Repeat Protein RF1A from Rice

Pentatricopeptide repeat protein (PPR) proteins are putative RNA-binding proteins which are particularly prevalent in terrestrial plants. Previous research has reported the great difficulty in purifying soluble PPR proteins in Escherichia coli, therefore hindering further study of their functions. In this paper, we report the use of the pMAL™ prokaryotic expression system to acquire a soluble expression of a PPR protein, RF1A from rice (Oryza sativa L.). After purification, we identified RF1A by ESI-TOF-MS/MS. We also made an estimation of its secondary structure using the circular dichroism spectroscopy. These results supported the bioinformatic prediction of helical-hairpin model about PPR proteins.

The editing sites in transcripts of functional genes of rice mitochondria

RNA editing exists extensively in the higher plant mitochondria, and is a required step for forming functional proteins. There may be some relationship between RNA editing and cytoplasmic male sterility (CMS), a kind of phenomenon that is attributed to mitochondrial genome mutations. The research materials used are the gametophytic male sterility line (A), maintainer line (B) and F1 hybrid (F1) of HL-type CMS rice. cDNAs and DNAs of atp6 and coxII have been obtained from A, B and F11 by PCR and RT-PCR. Comparing sequences of cDNAs and DNAs, 18 and 15 editing sites were found respectively in the transcripts of atp6 and coxII. A, B and F1 shared the same editing sites. RNA editing improves hydrophobicity and conservation of the predicted protein as compared with other organisms.

Discovery, utilization and perspective of Honglian cytoplasmic male sterile rice

Hybrid rice has been researched and utilized in China for 50 years. Three mainly cytoplasmic male sterile rice are wild abortive (WA), boro-II (BT), and Honglian (HL). Honglian (HL) type hybrid rice has made important contributions to the development of hybrid rice by combining scientific research, application research and industrial utilization. We identified Honglian cytoplasmic male sterile (HL-CMS) gene, orfH79 , which located downstream of atp6 in mitochondrial genome. CMS RNA, atp6-orfH79 , were translated into a cytotoxic protein, ORFH79. Data showed ORFH79 interacts with P61, which is a subunit of complex III of mitochondrial electron transfer chain. This interaction may impair energy production and leads to the accumulation of reactive oxygen species (ROS) in mitochondria, consequently resulting in abnormal pollen development. Furthermore, we cloned two restorer of fertility ( Rf ) genes, Rf5 and Rf6 , both of which are PPR genes. Neither RF5 nor RF6 binds to CMS RNA, atp6-orfH79 , directly. RF5 interacts with glycine rich protein 162 (GRP162) and RFC3 (restoration of fertility complex subunit 3) thus forming a restoration of fertility complex (RFC) to process CMS RNA. GRP162 can form homodimer and bind to CMS RNA with two binding sites directly. RCF3 is transported into the mitochondria as a membrane protein and functions as a scaffold protein for the assembly of the RFC complex. The RFC complex was detected with a size ranging from 400 to 500 kD. Moreover, Rf6 encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3 to 5. Results show RF6 is also targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS RNA, atp6-orfH79 , to restore the fertility of CMS line. To create more available germplasm in hybrid rice, we developed a molecular marker of orfH79 to screen wild rice and bred some novel CMS lines. We also developed molecular markers of Rf5 and Rf6 to breed novel stronger restore lines which contain two Rf genes. Consequently, we bred several combinations such as Honglian you 6, Yueyou 938, Luoyou 8 and Luoyou 9348 with high yield, good quality, multiple resistance and wide adaptation. HL hybrid rice has been widely planted in Yangzte river areas and South China with the yield of 10 to 12 tons per hectare. HL hybrid rice combinations also have been planted in Southeast Asian countries, such as Vietnam, Indonesia, Malaysia, Bangladesh and Phillippine. Most combinations exhibited 140% or more of yield compared with the local rice cultivars in Southeast Asian countries. HL hybrid rice even more is becoming the leading hybrid rice and priority choice in most of Asian countries and other Africa countries.

Construction of the Primary Physical Map of Rice Chromosome 12

A primary physical map of rice chromosome 12 was constructed using marker-based chromosome landing and chromosome walking. A BAC library from IR64 was screened using 84 RFLP markers, 4 STS markers and 6 microsatellite markers on chromosome 12 by colony hybridization and polymerase chain reaction (PCR) amplification. A total of 59 contigs consisting of 419 BAC clones including 5 single-clones were physically aligned on rice chromosome 12 with the largest BAC contig covering 855 kb. The whole physical map had a size of ∼16 Mb and covered about 52% of rice chromosome 12. This physical map will be certainly helpful for map-based gene cloning of agronomically and biological important genes and understanding the genome structure of the chromosome.

Studies of the energy release of mitochondria from sporophyte cytoplasmic male sterile rice by differential scanning calorimetry

The differential scanning calorimetric (DSC) curves of the mitochondria isolated from two varieties of sporophyte cytoplasmic male sterile and their fertile lines of Yie Bai and Ma Xie type rice have been determined. The curves show that the energy is released continuously as temperature rise to 70°C. Some thermodynamic and kinetic parameters of the energy release of the mitochondria have been obtained. The presented results showed that the mitochondria from cytoplasmic male sterile rice released more heat and they had higher energy barrier, less rate, and more complicated mechanism than that of their fertile lines in the energy release process.

Studies on molecular mechanism of rice cytoplasmic male sterility

Seedlings^ gene differential expression between rice cytoplasmic male sterile lines and their maintain lines of Zhenshan 97, Maxie and Congguang 41 was found by applying mRNA differential display. And there were different features in gene differential expression. This paper preliminarily analysed the result and further discussed the complex mechanism of interaction between nuclear and cytoplasmic gene in cytoplasmic male sterility.

Microcalorimetric study on the germination of seeds

The power-time curves of the germination of different rice and tree seeds have been studied by using a microcalorimeter set up by ourselves. Some characteristic values have been obtained from these curves in different thermogenesising stages of the germination of rice and tree seeds. These parameters can be used in comparison of character and activity for different seeds, moreover the effects of different strain germinating shape and temperature on the power-time curves of the germination of seeds have been discussed.