Ethylene response factor BnERF2-like (ERF2.4) from Brassica napus L.enhances submergence tolerance and alleviates oxidative damage caused by submergence in Arabidopsis thaliana

Ethylene response factor proteins play an important role in regulating a variety of stress responses in plants,but their exact functions in submergence stress are not well understood.In this study,we isolated BnE RF2.4 from Brassica napus L.to study its function in submergence tolerance.The expression of the BnE RF2.4 gene in B.napus and the expression of antioxidant enzyme genes in transgenic Arabidopsis were analyzed by quantitative RT-PCR.The expression of BnE RF2.4 was induced by submergence in B.napus and the overexpression of BnE RF2.4 in Arabidopsis increased the level of tolerance to submergence and oxidative stress.A histochemical method detected lower levels of H_2O_2,O^(·-)_2and malondialdehyde(MDA) in transgenic Arabidopsis.Compared to the wild type,transgenic lines also had higher soluble sugar content and higher activity of antioxidant enzymes,which helped to protect plants against the oxidative damage caused by submergence.It was concluded that BnE RF2.4 increased the tolerance of plants to submergence stress and may be involved in regulating soluble sugar content and the antioxidant system in defense against submergence stress.

Tissue-specific Expression of Acetolactate Synthase (ALS), Male Sterility-inducing Effect of Tribenuron-methyl and Its Effect on ALS Activity in Brassica napus L.

[Objectives]This study was conducted to provide a basis for the rapid identification of the drug spraying effect in early stage and the molecular mechanism of chemical hybridizing in Brassica napus L.[Methods]Quantitative RT-PCR analysis showed that ALS was constitutively expressed in various tissues of 096030,including flower buds,four floral organs (calyxes,petals,stamens and pistils),roots,stems and leaves.ALS was prominently expressed in leaves and was expressed weakly in the petals and stamens.The male sterility-inducing effects of tribenuron-methyl on such two Brassica napus L.varieties as Ningyou18 and 096030 were investigated.[Results]Plants were twice sprayed with 0.2 μg/ml tribenuron-methyl on leaves.The results showed that 8-10 ml of tribenuron- methyl was applied per plant for the first time at bolting stage with 1-2 mm flower buds on 15-20 cm inflorescence,and the second spray was performed with 8-10 ml of tribenuron-methyl per plant 10 d later.The results showed that the percentage of the full sterile plants reached 100%,which lasted for the whole flowering period,and the relative seed setting rate was only about 4%.Thus,this method could fullfill the requirement of hybrid seed production in field.The in-vivo enzyme activity of acetolactate synthase (ALS) was assayed using 2 mm buds collected 3 d after spray.The results showed that 0.2 μg/ml tribenuron-methyl inhibited ALS activity.The ALS activity of Ningyou 18 (CK) and Ningyou 18 (0.2 μg/ml) was 3.20 and 1.30 μmol/(mg·h),respectively,and the ALS activity of 096030 (CK) and 096030 (0.2 μg/ml) was 3.37 and 1.25 μmol/(mg·h),respectively.The relative enzyme activity of ALS in Ningyou18 and 096030 was 40.63% and 37.23%,respectively,both of which decreased significantly.[Conclusions]These results showed that the change of ALS activity may be used as an index for quickly identifying and predicting the chemical hybridizing effect of tribenuron-methyl.

Screening and Identification of Waterlogging Tolerance in Brassica napus Germplasm Resources

In order to evaluate,screen and identify waterlogging-tolerant Brassica napus resources and provide good germplasm materials for breeding of waterlogging-tolerant rape,608 B. napus germplasm materials from different sources were identified and screened for waterlogging-tolerant germplasms. The identification results showed that tested materials had dead seedling rates in the range of 0-100%,indicating that these rape germplasm materials varied extensively in waterlogging tolerance. Among the 608 materials,waterlogging-tolerant materials( grade I,relative dead seedling rate≤21%) accounted for 25. 49%; non-tolerant materials( grade Ⅳ + V,dead seedling rate≥61%) accounted for 29. 43%; and other materials had waterlogging tolerance between the two. The identification results of waterlogging tolerance in the 349 rape germplasm resource from the upper,middle and lower reaches of Yangtze River and Huang-Huai Basin showed that materials from the lower Yangtze River had the lowest average dead seedling rate of 38. 8%,which was remarkably lower than materials from other ecological regions,and waterlogging-tolerant materials among them accounted for 33. 6%,which was also remarkably higher than materials from other ecological regions. It could thus be seen that materials from the lower Yangtze River have stronger waterlogging tolerance than those from other ecological regions overall. In this study,24 highly-tolerant B. napus germplasms( dead seedling rate < 1%) were selected from the 608 B. napus germplasm materials,which could be used for breeding of waterlogging-tolerant rape and related fundamental research.

Cloning of CRABS CLAW Gene from Brassica napus and Construction of Its RNA Interference Expression Vector

CRABS ClAW （CRC） is a member of the YABBYA transcription factor gene family that plays an important role in floral organ development of plants. This study aimed to further investigate the regulatory function of CRC transcription factor in the development of floral organs of rape （Brassica napus L. ）. A 580 bp fragment of CRC gene was cloned by RT-PCR from total RNA of buds of rape cultivar Ningyou No. 10 to construct an inverted repeated expression cassette of CRC gene using intermediate vector pHturieane. Firstly, CRC gene fragment was positively inserted into the 5＇ end of a spliceable intron and negatively inserted into the 3＇ end of the intron. Subsequently, CaMV35S promoter sequence and inverted repeated expression cassette of CRC gene were transferred into pUC18 multiple clone site of binary expression vector pCAMBIAI1390. The constructed interference expression vector was named pA6-CRCi, which was further confirmed by restriction enzyme digestion and sequencing.

Indoor Propagation of Transgenic Glyphosate-tolerant Rapeseed (Brassica napus L.) and Transfer Breeding for Herbicide Tolerance

[Objectives] This study was conducted to investigate the possibility of screening for herbicide resistance in the early stage of rapes growth. [Methods] Eight treatments were set for a herbicide concentration experiment. [Results] Rape sprouts were highly sensitive to the herbicide glyphosate, and even a very low concentration of glyphosate solution(187 mg/L) was sufficient to kill most of the rape seedlings, while the genetically modified herbicide-tolerant rape could tolerate higher concentrations of the herbicide. Low concentrations of glyphosate could be used for screening at the sprout stage, and the results of herbicide-tolerance screening were consistent with PCR testing. [Conclusions] This study lays a technical and material foundation for accelerating the cultivation of new herbicide-resistant rape varieties suitable for rapeseed production areas in the lower reaches of the Yangtze River.

Detection of Quantitative Trait Loci for Yield and Drought Tolerance Traits in Soybean Using a Recombinant Inbred Line Population

To investigate the genetic basis of drought tolerance in soybean （Glycine max L. Merr.） a recombinant inbred population with 184 F2：7：11 lines developed from a cross between Kefengl （drought tolerant） and Nannong1138-2 （drought sensitive） were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci （QTLs） were identified： 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and Dlb, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.

Mapping QTLs for seed yield and drought susceptibility index in soybean (Glycine max L.) across different environments

Drought stress has long been a major constraint in maintaining yield stability of soybean （Glycine max （L.） Merr.） in rainfed ecosystems. The identification of consistent quantitative trait loci （QTL） involving seed yield per plant （YP） and drought susceptibility index （DSI） in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F2：7：11 lines from a cross of Kefengl and Nannong1138-2 was studied under water-stressed （WS） and well-watered （WW） conditions in field （F） and greenhouse （G） trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identi- fied. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits re- lated to yield and other agronomic traits. One QTL on molecular linkage group （MLG） K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.