Molecular Detection And Cold Tolerance Analysis Of Transgenic Cotton With Insect Antifreeze Protein Gene Mpafp149

Low temperature and cold damage is one of the most important abiotic stressfactors that affect the growth and yield of agricultural crops. Many economicallyimportant crops, such as cotton, soybean, corn and rice are very susceptible to chilling,and can not survive in cold temperatures. Cotton is one of the world’s mosteconomically important crops and renewable resources. Xinjiang cotton areas havesuperior natural conditions, large-scale production accounting for about1/3of thecountry’s total output of cotton, but the frequent occurrence of cold weather during thespring season in these areas causes serious injury or death of cotton plants. The lowtemperature will cause serious damage or death to cotton knot peach, cleft bell and bollopening stage of the plants, which greatly affect the yield and quality of cotton.Using traditional breeding methods to improve plant cold hardiness is oftentime-consuming and laborious and difficult to modify single trait; depends largely onthe existing varieties. Genetic engineering is a relatively fast and accurate way toimprove plant cold hardiness. Antifreeze protein enables many organisms frominvertebrates to bacteria to survive sub-zero environments. The thermal hysteresisactivity of insect antifreeze proteins is10-100times higher than most other organismsthat have been found to antifreeze protein activity. This makes insect antifreeze proteingreat potential and advantages in improving plant cold hardiness through genetictransformation process.In the present work, an insect antifreeze protein gene Mpafp149was transformedinto the north Xinjiang cotton-growing cultivars by pollen tube pathway to decreasecold and frost damage to cotton plant caused by low temperatures during early springand early autumn. The transgenic cotton offspring generated in2010-2012werescreened by several molecular detections, including PCR, RT-PCR, Southern blot,Western blot and other methods. The cold hardiness of the transgenic plants were testedafter cold treatment, including phenotypic observation, determination of relative conductivity, content of malondialdehyde (MDA) and proline.PCR and PCR-Southern blot results showed that Mpafp149have been passed to theoffspring of transgenic cotton. The number of positive PCR plants in2011.7MNS T2and MNS T1transgenic cotton is306, of which99is in T2generation,207is in T1generation. The positive rate was60%and53%, respectively. The number of kanamycinresistant plants in2012.5Lab2T3generation transgenic cottons is87, NPT IIPCR-positive-number is100, Mpafp149PCR-positive number is86, and the positiverate was82%,94%and81%, respectively. RT-PCR results showed that the insectantifreeze protein gene has been expressed at transcriptional level in transgenic cottons.RT-PCR positive-number of2012.3Lab1T3transgenic cotton is13, and the positiverate of30%.Western blot showed that the expected protein bands abou (10kDa)wereobserved in the T3generation transgenic lines8-3,8-4hybridization results, whichfurther illustrated the expression of heterologous insect antifreeze protein MpAFP149inT3generation transgenic cotton plants.T4generation transgenic lines T3-12, T3-14,174-5were treated in2℃for24,48,and72h. Phenotypic observation indicated that the transgenic cotton plants showed lesscold damage than the wild type plant. After cold treatment for72h, the relativeconductivity of wild-type cotton, T3-12and T3-14were80%,45%and57%,respectively; The increment of MDA content were21.9,16.8and15.7μmol/g,respectively. These results showed that the changes of relative conductivity and MDAcontent for transgenic plants were less than wild-type cotton, which reflected that theplasma membrane of transgenic cotton was suffered less chilling injury by lowtemperature treatment. In addition, after cold treatment for72h, proline content of thewild-type cotton, T3-12and T3-14were0.4,0.85and0.81μg/g, respectively, indicatingthat the transgenic plants produce more proline to improve the resistance to lowtemperature stress. Taken these results together, it shows that the cold tolerance oftransgenic cotton was improved to certain extent by transferring Mpafp149.Label-free porous silicon (PSi) biosensor is a promising fast and efficient detection platform for biological molecules. It has been widely used for the detection of variousbiomolecules. Detection of antifreeze protein based on Single-layer porous siliconprotein biosensor showed that significant red shifts were observed for all transgenicfrost-resistant cotton lines, except transgenic line2, compared with non-transgeniccotton plants. This result indicated that the exogenous antifreeze gene has beenexpressed on the protein level of the transgenic cotton. The detection limit of the PSibiosensor is0.0027mg·ml-1.In addition, we took advantage of the multi-photonic crystal structure of poroussilicon sensor to detect the hybridization between antifreeze gene probe and itscomplementary DNA fragments. The result showed that the complementaryhybridization between these DNAs can be detected. The ultimate detection limit is0.0213μM. These PSi biosensors provided a new idea for transgenic screening and havethe potential to become a stable and sensitive platform for detecting of large-scaletransgenic plants screening in the field.