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1.
To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3′end of the second intron of Arabidopsis
AGAMOUS and CaMV 35S (−60) minimal promoter [AG-I-35S (−60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (−60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering.
Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination.
When controlled by AG-I-35S (−60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents
the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (−60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy
for interception of transgene escape and has other potentially commercial use for transgenic engineering. 相似文献
2.
A potential problem in the field release of transgenic plants is the spread
of foreign gene products via pollen. Therefore, the use of the tomato
pollen-specific lat52 gene promoter was investigated
as a means of targeting antisense RNA to pollen without affecting transgene
expression elsewhere in the plant. A transgenic tobacco line T115, which
showed GUS expression in pollen, leaves and roots were retransformed with a
construct containing the pollen-specific lat52
promoter driving the GUS encoding uid A gene in
antisense orientation. From 24 independent transformants obtained, 19
showed a significant reduction in pollen GUS activity. Of these lines, four
showed a reproducible antisense effect in pollen in the next generation,
while it was shown in one line that GUS activity in leaves and roots was
also unaffected. To ascertain the effectiveness of the antisense strategy
to downregulate very high levels of pollen expression, a
lat52-gus antisense construct was introduced into
tobacco lines containing lat52-gus, which had pollen
GUS activity of up to 250 times greater than in line T115. Results showed
that 30 out of 34 independent lines exhibited a significant antisense
effect in pollen, confirming the effectiveness of pollen-targeted antisense
strategy to reduce undesirable expression in pollen independent of
expression level in pollen. 相似文献
3.
4.
Gene containment technologies that prevent transgene dispersal through pollen, fruit and seed are in immediate demand to address
concerns of gene flow from transgenic crops into wild species or close relatives. In this study, we isolated the enhancer
element of Arabidopsis
AGAMOUS that drives gene expression specifically in stamens and carpels. By fusing this AG enhancer to a minimal 35S promoter fragment, two tissue-specific promoters, fAGIP and rAGIP in forward and reverse orientations, respectively, were created and fused to the GUS reporter. Transgenic Arabidopsis plants harboring either fAGIP::GUS or rAGIP::GUS displayed similar GUS expression specifically in carpel and stamen tissues and their primordial cells. To test their utility
for engineering sterility, the promoters were fused to the Diphtheria toxin A (DT-A) gene coding for a ribosome inactivating protein as well as the Barnase gene coding for an extracellular ribonuclease, and tested for tissue-specific ablation. Over 89% of AGIP::DT-A and 68% of AGIP::Barnase transgenic plants displayed specific and precise ablation of stamens and carpels and are completely sterile. These transgenic
plants showed normal vegetative development with prolonged vegetative growth. To evaluate the stability of the sterile phenotype,
16 AGIP::DT-A lines underwent two consecutive cutback generations and showed no reversion of the floral phenotype. This study demonstrates
a simple, precise and efficient approach to achieve absolute sterility through irreversible ablation of both male and female
floral organs. This approach should have a practical application for transgene containment in ornamental, landscaping, and
woody species, whose seeds and fruits are of no economic value. 相似文献
5.
A novel 407 bp nucleotide sequence NTPp13 was isolated from tobacco (Nicotiana tabacum L.) by PCR, its structure and function were characterized. The NTPp13 sequence was highly homologous with the pollen-specific expression promoter Zm13 from maize (Zea mays L.) and contained some key motifs which controlled pollen-specific expression. The NTPp13 was fused to the β-glucuronidase (GUS) reporter gene and transferred into tobacco. Analysis of the transgenic plants revealed
that this putative promoter fragment was sufficient to direct GUS expression specifically in the anther, exactly in the pollen
and pollen tube, and that GUS activity reached the maximum at the stage of pollen grain began to separate. Further study showed
that the expression of NTPp13 sequence at pollen was stable at the range of temperature measured. These data suggested that the NTPp13 sequence was likely the essential element of promoter region of an unknown pollen-specific gene from tobacco. 相似文献
6.
Cao Bihao Wei Xiaosan Lei Jianjun Xiao Xiou Chen Qinhua 《Plant Cell, Tissue and Organ Culture》2012,111(2):163-172
Production of hybrid seeds and pursuing heterosis breeding of many crops have been accomplished using male sterile lines. However, not all crops have valuable male sterile lines due to instability of male sterility and absence of a restorer system. In this study, male sterile lines have been induced using a two-component system. The extracellular ribonuclease Barnase was cleaves into two inactive yet complementary fragments, designated as ??Bn-5?? and ??Bn-3??. Both components were controlled by a TA29 promoter. They were transferred into the tomato inbred line ??Yellow tomato?? by Agrobacterium method. Southern blotting identified that 11 transgenic Bn-5 plants (T0) and 10 transgenic Bn-3 plants (T0) were obtained. The vegetative phenotypes of all T0 plants were similar to wild-type, and they were capable of producing viable pollen grains and normal fruit with seeds, indicating that Barnase had lost its function after it being split two partial fragments. After self-pollination, homozygous progenies (T1) of transgenic Bn-5 and Bn-3 plants were chosen to cross each other, Barnase could be reconstituted and co-expressed in the same cell, which caused the hybrid plants to produce collapsed pollen grains with no viability and thus100?% male sterile plants were obtained. Stamens of male sterile plants were shorter than those of the wild type plants. PCR detection demonstrated that all male sterile plants contained Barnase, but male fertile plants did not. The male sterile plants were crossed with the male fertile inbred lines, and the result showed that hybrid (F1) plants were capable of producing normal fruit with seeds, and their pollen grain fertility was restored. The co-segregation ratio of Bn-5 and Bn-3 fragments showed 1:1 among hybrid plants. In conclusion, the results verified that the male sterility could be generated by two component system and be used in hybrid seed production. The F1 between the male sterile plant and the inbred line showed heterotic comparing to both parents. This system needs not breed restoration line. 相似文献
7.
The superior performance of F1 hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient
system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference
(RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with
a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis
stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed
pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants
with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size
of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when
cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These
results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines
for producing hybrid seed. 相似文献
8.
Hans-Hubert Kirch Yi-Qin Li Ursula Seul Richard D. Thompson 《Sexual plant reproduction》1995,8(2):77-84
Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S
2
-RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S
2
RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S
2
-RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S
2
polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S
2
-RNase protein in pollen. 相似文献
9.
Ling Chen Yingjie Miao Cheng Wang Peipei Su Tianheng Li Rong Wang Xinglong Hao Guangxiao Yang Guangyuan He Chunbao Gao 《Plant Molecular Biology Reporter》2012,30(6):1426-1432
PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the ??-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4?kb fragment of the promoter was enough to confer pollen-specific expression. 相似文献
10.
Ji-tao Zou Xiao-yan Zhan Hen-ming Wu Hon g Wang Alice Y. Cheung 《American journal of botany》1994,81(5):552-561
Pollen development requires a large number of genes expressed in both sporophytic and gametophytic tissues. We have isolated a pollen-specific gene, PS1, from rice. PS1 is a unique gene in the rice genome and encodes a 164 amino acid long protein. RNA blot analysis shows that PS1 mRNAs accumulate specifically in rice anthers. When introduced into rice tissues by microprojectile bombardment, the PS1 promoter drives expression of a marker gene, β-glucuronidase, specifically in rice pollen. The PS1 gene and the deduced amino acid sequence of the PS1 protein share significant levels of homology with another monocot pollen-specific gene—the maize Zm-13 gene and its deduced protein, respectively. PS1 also shows some homology with the dicot tomato anther-specific gene LAT-52. Interestingly, the structure of the PS1 gene is more similar to that of the LAT-52 gene than to Zm-13. The coding regions of both PS1 and LAT-52 are interrupted by a single intron, and the positions of the introns are conserved in these genes. Moreover, there is considerable sequence homology in the introns of the PS1 and LAT-52 genes in regions immediately upstream of the 3' splice sites. The upstream regulatory sequences of the PS1 gene show several regions of homology with other pollen- or anther-specific genes from a number of plant species. The conservation of coding sequences of PS1 from rice, Zm-I3 from maize, and LAT-52 from tomato suggests a functional conservation of their gene products. Similarities in the regulatory regions of PS1 and other anther- or pollen-specific genes among monocotyledonous and dicotyledonous species indicate that at least some regulatory features controlling gene expression in male reproductive tissues are conserved. This is supported by the preservation of pollen-specific expression from the rice PS1 promoter when it is introduced into tobacco plants by Agrobacterium Ti plasmid-mediated transformation. 相似文献
11.
Construction of a multicontrol sterility system for a maize male‐sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD‐finger transcription factor 下载免费PDF全文
Yan Zhou Liwen Xu Wen Fang Shensi Liu Shuangshuang Liu Taotao Zhu Jinping Li Liqun Rao Jiuran Zhao Xiangyuan Wan 《Plant biotechnology journal》2018,16(2):459-471
12.
Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease 下载免费PDF全文
Reginald J. Millwood Hong S. Moon Charleson R. Poovaiah Balasubramaniam Muthukumar John Hollis Rice Jason M. Abercrombie Laura L. Abercrombie William Derek Green Charles Neal Stewart Jr 《Plant biotechnology journal》2016,14(5):1281-1290
Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species. 相似文献
13.
Key message
BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology.Abstract
We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes’ extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility. 相似文献14.
Xiaoyan Dai Jingjuan Yu Jinxia Ma Guangming Ao Qian Zhao 《Plant Growth Regulation》2007,52(3):229-239
We previously identified a 0.7 Kb cDNA fragment of Zm401, a novel pollen-specific gene in maize (Zea mays). However, little information is known about the function of Zm401 in pollen development. The full-length of Zm401 cDNA was amplified by 5′ RACE and 3′ RACE and both sequence analysis and in vitro translation of Zm401 showed that it belonged to an mRNA-like non-coding gene. To analyze its possible biological roles in pollen development,
the Zm401 cDNA was overexpressed in transgenic maize under the control of a pollen specific promoter Zm13 or a CaMV 35S promoter. RT-PCR and RNA gel blot analysis indicated that the expression level of Zm401 in leaves and anthers of transgenic plants was much higher than that of non-transformants. Compared with the non-transformed
maize, transgenic maize showed distinct phenotypes, such as abnormal tassels and degenerate anthers. The histological observation
showed that the development of pollen grains and anthers in transgenic plants were abnormal. These abnormalities include delayed
degradation of tapetum, asynchronous fusion of pollen sacs, and aborted pollen grain development. Furthermore, the pollen
viability in six transgenic plants ranged from 1.24% to 6.63%. The reduced pollen viability cosegregated with the transgene
in a selfed progeny. These results suggest that Zm401 is involved in the regulation of pollen development. This article demonstrated Zm401, as a non-coding RNA, plays an essential role in pollen development.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
基因工程雄性不育烟草及其温度敏感性 总被引:23,自引:1,他引:23
将含有抗溴苯腈基因bxn和雄性不育基因的重组载体pTA29-Barnase/bxn导入农杆菌(Agrobacterium tumefaciens)后转化烟草(Nicotiana tabacum L.),得到33个转基因植株。在27℃/23℃培养的16株中,有7株表现部分不育,另9株全部可育。而在20℃/15℃培养的17株中,12株表现不育,5株表现部分不育。部分不育的植株上同时开放可育花朵和不育花朵,不过其中的不育花朵中的花粉萌发活力很低。将在20℃/15℃条件下表现不育的12个不育株从20℃/15℃温室移至27℃/23℃温室后30d左右,其中9株表现程度不同的育性恢复现象:5株表现部分可育,另4株表现完全可育;但仍有3株表现雄性不育。雄性不育花朵的花丝变短,花药皱瘪,不散粉。细胞学观察证明,转基因植株的花药绒毡层降解提早于四分体时期,至单核小孢子时期降解殆尽。 相似文献
16.
17.
Arun Viswanathan Boney Kuriakose Shantharam Bharadwaj George Thomas 《Plant Molecular Biology Reporter》2011,29(4):825-834
Expression of many proteinases has been documented during anther development. Although their roles are not completely understood,
their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such
an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid
seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed
the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged
tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen
production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during
anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases
in anther development still has to be elucidated. 相似文献
18.
Bayer Martin Hess Dieter 《Molecular breeding : new strategies in plant improvement》2005,15(2):193-203
Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T0-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained. 相似文献
19.
20.
FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type
tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased
levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased
FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing
tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants
from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated
GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue
culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast
size and number. 相似文献