Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a Better Gene-Silencing Strategy to Study Gene Functions in Rice

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

show abstract

“…The TMS5as vector was constructed as previously described50. An antisense fragment from TMS5 was amplified and cloned into pYLox vector15.…”

Section: Methodsmentioning

confidence: 99%

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System

Zhou¹,

et al. 2016

Sci Rep

Self Cite

194

126

View full text Add to dashboard Cite

show abstract

“…The cDNA levels of target genes were normalized to the internal standard genes OsActin1 and OsEF1-a. RNA blotting was performed as previously described 42 . The hybridization signals were visualized using a Typhoon 9000 System (GE, USA).…”

Section: Subcellular Localizationmentioning

confidence: 99%

“…Total protein was extracted from rice panicles grown at different temperatures. Immunoblot analysis was performed using Li's methods 42 . The complementary sequences corresponding to tRNAs were used as probes labelled with g-32 P-ATP using T4 polynucleotide kinase.…”

Section: Subcellular Localizationmentioning

confidence: 99%

RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice

et al. 2014

Self Cite

View full text Add to dashboard Cite

Thermosensitive genic male-sterile (TGMS) lines, which are male-sterile at restrictive (high) temperatures but male-fertile at permissive (low) temperatures, have been widely used in breeding two-line hybrid rice (Oryza sativa L.). Here we find that mutation of thermosensitive genic male sterile 5 (tms5) in rice causes the TGMS trait through a loss of RNase Z S1 function. We show that RNase Z S1 processes the mRNAs of three ubiquitin fusion ribosomal protein L40 (Ub L40 ) genes into multiple fragments in vitro and in vivo. In tms5 mutants, high temperature results in increased levels of Ub L40 mRNAs. Overaccumulation of Ub L40 mRNAs causes defective pollen production and male sterility. Our results uncover a novel mechanism of RNase Z S1 -mediated Ub L40 mRNA regulation and shows that loss of this regulation produces TGMS in rice, a finding with potential applications in hybrid crop breeding.

show abstract

“…To prepare the overexpression construct of P/TMS12-1, a 375-bp fragment (PA-0.37) of P/TMS12-1 was amplified and inserted into the Hind III/Spe I sites downstream of the promoter of maize ubiquitin (Pubi) in a pCAMBIA-based vector. These constructs were transformed into the Agrobacterium tumefaciens strain EHA105 and were used for rice transformation by a previously described method [65].…”

Section: Preparation Of Binary Constructs and Rice Transformationmentioning

confidence: 99%

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

et al. 2012

Self Cite

View full text Add to dashboard Cite

Photoperiod-and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo-or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions. Keywords: rice; PGMS; TGMS; noncoding RNA; small RNA Cell Research (2012) IntroductionAsian cultivated rice (Oryza sativa L.) is one of the world's most important crops, meeting the stable food demand of more than half of the global population. The development of hybrid rice is a major approach for increasing the yield potential of rice, since hybrid rice varieties have about 20% or more yield advantage over improved inbred varieties [1]. Hybrid rice technologies are mainly based on the three-line and two-line systems. The three-line system uses cytoplasmic male sterility lines, maintainer lines and restorer lines [2,3]. The twoline hybrid rice is based on the discovery and application of environmentally sensitive genic male sterile (EGMS) lines, which serve as both the male sterile lines and maintainer lines under different environmental conditions [4]. Thus, the two-line hybrid rice system is an important innovation for the better exploitation of hybrid vigor (heterosis). Compared with the three-line system, the advantages of the two-line system include a wider range Rice PGMS and TGMS caused by a point mutation in a noncoding RNA 650 of germplasm resources used as breeding parents, better grain quality and higher yields, and benefiting from simpler procedures for breeding and hybrid seed production [5...

show abstract

Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a Better Gene-Silencing Strategy to Study Gene Functions in Rice

Cited by 32 publications

References 39 publications

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System

RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Contact Info

Product

Resources

About