Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the c...Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.展开更多
Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor....Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.展开更多
Citrus is mainly oriented to fresh consumption,and harvesting is usually performed manually.However,the high cost of harvesting and the low availability of labour can compromise the profitability of the crop when it i...Citrus is mainly oriented to fresh consumption,and harvesting is usually performed manually.However,the high cost of harvesting and the low availability of labour can compromise the profitability of the crop when it is destined to juice industry.The development of citrus mechanical harvesting for industrial processing is conditioned to reach a high fruit removal efficiency,with a reduced damage to both fruit and trees.The current machines available are very large,requiring extensive plantations with long rows of trees to be efficient.In this study,two lateral canopy shakers equipped with a catch frame were evaluated to harvest independently both sides of the hedge on intensive citrus plantations with the main objective of determining their performance and feasibility.The lateral canopy shakers tested were three tractor-drawn machines,one commercial machine and two prototypes.The tested machines reached a mean value of 78%of fruit removal.Besides,the prototypes,equipped with a catch frame,were able to recover a mean value of 70%of yield.Although the results were promising,for achieving an efficient result,the application of this harvesting technology still requires a process of improvement,and the adaptation of both the machine and the plantation.The machines should reduce the amount of post-harvest ground fruit(5.9%-10.4%).Tree damages generated by the contact of the catch frame with the trunk and the metal rods with main branches were the most relevant.Therefore,it is still necessary to increase the ground speed of the machinery and improving the design of the rods,regulating the rod penetrating deep in the canopy to improve the fruit recovery and limit the damage caused to the trees.展开更多
文摘Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.
基金supported by the Fundamental Research Funds for the Central Universities (Grant No. XDJK 2018B016)the National Natural Sciences Foundation of China (Grant No. 31972393)+1 种基金he earmarked fund for China Agriculture Research System (Grant No. CARS-26)the Natural Science Foundation of Chongqing (Grant No. cstc2020jcyj-msxmX1064)。
文摘Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.
基金This work has been supported by research project RTA2014-00025-C05-03 from the National Institute for Agricultural and Food Research and Technology(INIA,Spain)financed by FEDER funds.Authors wish to acknowledge the support of Spanish Ministry of Economy and Competitiveness(Pre-commercial public procurement Mecaolivar),Cítricos del Andévalo S.L.for the support and funding received for the adaptation of the prototypes and Moresil S.L.and Maqtec Inc.for the developed prototypes.
文摘Citrus is mainly oriented to fresh consumption,and harvesting is usually performed manually.However,the high cost of harvesting and the low availability of labour can compromise the profitability of the crop when it is destined to juice industry.The development of citrus mechanical harvesting for industrial processing is conditioned to reach a high fruit removal efficiency,with a reduced damage to both fruit and trees.The current machines available are very large,requiring extensive plantations with long rows of trees to be efficient.In this study,two lateral canopy shakers equipped with a catch frame were evaluated to harvest independently both sides of the hedge on intensive citrus plantations with the main objective of determining their performance and feasibility.The lateral canopy shakers tested were three tractor-drawn machines,one commercial machine and two prototypes.The tested machines reached a mean value of 78%of fruit removal.Besides,the prototypes,equipped with a catch frame,were able to recover a mean value of 70%of yield.Although the results were promising,for achieving an efficient result,the application of this harvesting technology still requires a process of improvement,and the adaptation of both the machine and the plantation.The machines should reduce the amount of post-harvest ground fruit(5.9%-10.4%).Tree damages generated by the contact of the catch frame with the trunk and the metal rods with main branches were the most relevant.Therefore,it is still necessary to increase the ground speed of the machinery and improving the design of the rods,regulating the rod penetrating deep in the canopy to improve the fruit recovery and limit the damage caused to the trees.
